Immunology Reports
Enhanced Survival During Experimental Listeria monocytogenes Sepsis in Neonatal Mice Prophylactically Treated With Th1 and Macrophage Immunoregulatory Cytokines and Mediators Mark B. Geyer, MD,* Kavita Radhakrishnan, MD,†‡ Carmella Van de Ven, MA,‡ Mandhir S. Suri, MD,§ Janet Ayello, MS,‡ and Mitchell S. Cairo, MD*‡¶‖**†† Background: Impairments in T-cell and macrophage-mediated host defense lead to increased infection-related morbidity and mortality in neonates, partly because of immaturity in T helper (Th)1 function. Listeria monocytogenes (Lm) is an intracellular pathogen disproportionately causing severe disease among neonates and the immunocompromised. Intact macrophage and Th1-mediated immune responses are critical for Lm clearance. We and others have previously demonstrated downregulation of Th1 and macrophage immunoregulatory cytokines in cord blood versus adult peripheral blood. We sought to determine whether therapeutic or prophylactic single agent or combination recombinant murine interleukin (rmIL)12, rmIL-18, recombinant murine macrophage-colony stimulating factor (rmM-CSF), recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) and recombinant murine interferon (rmIFN)-γ would enhance survival during experimental neonatal Lm sepsis. Methods: A 90% lethal dose (LD90) of Lm was established in C57/BL/6 neonatal mice. rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ were administered singly or sequentially, before or after LD90 Lm inoculation; ampicillin was administered 24 hours after inoculation. Results: Therapeutic doses of rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ as single agents and sequential therapy with rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were not associated with increased survival. However, prophylactic single doses of rmIL-12, rmIL-18, rmM-CSF and rmIFN-γ and prophylactic sequential doses of rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were associated with significantly enhanced survival compared with ampicillin alone. Conclusions: These data suggest prophylactic administration of macrophage and Th1 immunoregulatory cytokines can potentially overcome deficits in neonatal immunity to protect against Lm. Key Words: Listeria monocytogenes, neonatal, cytokine, interleukins, host defense (Pediatr Infect Dis J 2014;33:e330–e337)
Accepted for publication May 29, 2014. From the *Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; †Department of Pediatrics, University of California San Francisco, San Francisco, CA; ‡Department of Pediatrics, New York Medical College, Valhalla, NY; §Department of Pediatrics, Children’s Hospital and Health Center, San Diego, CA; and Department of ¶Medicine, ‖Pathology, **Microbiology and Immunology, and ††Cell Biology and Anatomy, New York Medical College, Valhalla, NY. Presented in part at the Pediatric Academic Societies Meeting, May 2003, Seattle, WA, and the American Society of Pediatric Hematology/Oncology Meeting, April–May 2004, San Francisco, CA. New York Medical College (PI: M.S.C.) received a grant from the Pediatric Cancer Research Foundation (Irvine, CA) in part to support this work. The authors have no other funding or conflict of interest to disclose. Address for correspondence: Mitchell S. Cairo, MD, Departments of Pediatrics, Medicine, Pathology, Microbiology and Immunology, and Cell Biology and Anatomy, New York Medical College, 19 Skyline Drive, Room IN-D12, Valhalla, NY 10595. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 0891-3668/14/3312-e330 DOI: 10.1097/INF.0000000000000442
e330 | www.pidj.com
L
isteria monocytogenes (Lm), a small, facultative anaerobe, gram-positive bacillus, can cause severe foodborne illness and remains a major cause of life-threatening bacterial infection in neonates. It is estimated that 2500 cases of severe listeriosis occur in the United States annually, mostly among pregnant women, neonates and immunocompromised adults, causing some 2300 hospitalizations and 500 deaths.1,2 Nonperinatal listeriosis-related deaths have decreased in the past 2 decades, presumably secondary to closer monitoring of food manufacturing.3 However, despite the widespread availability of antibiotic treatments for listeriosis, reported case fatality rates for neonates have ranged from 40% to 56%.4 Neonatal listeriosis may take the form of bacteremia and/or CNS infections. Early-onset Listeria sepsis is generally associated with maternal infection, premature delivery and a high case fatality rate. Lm is readily phagocytosed by host macrophages and other nucleated cells and incorporated into a phagolysosome. Lm subsequently lyses the phagolysosome through the actions of a secreted bacterial hemolysin (listeriolysin O) and enters the cytosol, where it directs polymerization of host-cell actin through the ActA transmembrane protein to achieve motility within the cytosol and spread from cell to cell. Because Lm is an intracellular pathogen, intact macrophage and cell-mediated immunity are critical to successful host defense.5 In adult mice, increased production of interferon (IFN)-γ by Lm-infected mice leads to macrophage activation and clearance of Lm infection.6,7 Colony stimulating factor (CSF)-1 and macrophage (M)-CSF regulate differentiation, proliferation and survival of macrophages and serve as a macrophage chemoattractants, and interleukin (IL)-12 and IL-18 act synergistically to induce IFN-γ production by natural killer (NK) and T helper (Th)1 cells during the host immune response to Lm infection. Neonates are characterized by their increased susceptibility to infection, particularly when T-cell and/or macrophage-mediated immune responses are required, by reduced responses to vaccineinduced antigenic stimulation and by the highest rates of infectious mortality in the human lifespan.8 In part, intrinsic bias against Th1 immune responses in the neonate has been implicated in increased susceptibility to bacterial infection.9,10 IFN-γ protein production and gene expression from stimulated umbilical cord blood (UCB) versus adult peripheral blood (PB) mononuclear cells (MNC) are significantly decreased.11–13 We have previously demonstrated significant downregulation of IL-12, IL-18, M-CSF and granulocyte macrophage (GM)-CSF mRNA production and decreased posttranscriptional stability in stimulated UCB versus adult PB MNC.14–17 Given the deficits observed in Th1 and macrophage immunoregulatory cytokines in neonatal cord blood and the mortality observed in neonates despite prompt antibiotic therapy in Lm sepsis and the observed roles of IL-12, IL-18 and M-CSF in the adult murine host response to Lm infection, we hypothesized that prophylactic administration of these cytokines might have a protective effect in Lm sepsis in a neonatal mouse model. In this study, we therefore assessed the effects on survival of exogenous administration of prophylactic doses of Th1 and macrophage
The Pediatric Infectious Disease Journal • Volume 33, Number 12, December 2014
The Pediatric Infectious Disease Journal • Volume 33, Number 12, December 2014
immunoregulatory cytokines and mediators, singly and sequentially, including IL-12, IL-18, M-CSF and GM-CSF in neonatal mice during experimental Lm sepsis.
MATERIALS AND METHODS Animals Adult wild-type (WT) C57BL/6 mice (20–25 g) and litters of C57BL/6 WT neonatal mice (≤24 hours old; 1.2–1.7 g) were used for this study. All animals were obtained from Jackson Laboratories, Bar Harbor, ME. Mothers of C57BL/6 litters were received 1 week before delivery. All animals were housed in the vivarium at Columbia University and maintained at constant room temperature with water and rodent feed ad libitum. Neonatal mice were pooled and then randomly split as equally as possible to each of the mothers; this was done in order to avoid any litter bias in the experiments. Approval for this study was obtained from the Institutional Animal Care and Use Committee at Columbia University.
Organism Lm (ATCC 7644, Manassas, VA) was grown in brain heart infusion to logarithmic phase, aliquoted, and stored at –70°C until use. Bacterial suspensions of 3 × 105 – 3 × 108 organism/g body weight/100 μL were prepared in phosphate buffered saline (PBS)/ human serum albumin (HSA) for 90% lethal dose (LD90) and cytokine studies.
Cytokines Recombinant murine (rm) IL-12 (0.01 μg/g body weight), rmIL-18 (0.1 μg/g body weight), rmIFN-γ (5 μg/pup), rmM-CSF (5 μg/kg body weight) and rmGM-CSF (3 μg/kg body weight) (R & D Systems Inc, Minneapolis, MN) were used in the study. All cytokines were purchased from R&D Systems, Inc. (Minneapolis, MN). Endotoxin levels in each batch were confirmed to be 10%) were euthanized and considered nonsurvivors.
Antibiotic Ampicillin (Amp) 100 mg/kg subcutaneous q12h was administered 24 hours after Lm injection to specific subgroups of Lm-infected animals.
Therapeutic Single or Repeated Dose of Cytokines Litters of neonatal mice (n = 10–15) received a dose of rmIL-12, rmIL-18, rmIL-12 + rmIL-18, rmIFN-γ, rmM-CSF or rmGM-CSF at 6 hours (single dose) or 6 hours and 24 hours (repeated dose) after infection with the LD90 dose of Lm, with or without Amp. Control animals received PBS/normal saline (NS) in lieu of cytokines, with or without Amp (Tables 1–3). Mortality was monitored and recorded daily for up to 7 days. © 2014 Lippincott Williams & Wilkins
Cytokine Prophylaxis in Listeria Sepsis
Therapeutic Sequential Doses of Cytokines Litters of neonatal mice (n = 10) received sequential doses of cytokines consisting of rmM-CSF at 2 hours, rmIL-12 and/or rmIL-18 at 6 hours and rmIFN-γ at 18 hours after the administration of the LD90 dose of Lm with or without Amp. Control animals received PBS/NS in lieu of cytokines with or without Amp (Tables 1–3). Mortality was monitored and recorded daily for 7 days.
Prophylactic Single Dose of Cytokines Litters of neonatal mice (n = 10–12) received a single dose of rmIL-12, rmIL-18, rmIL-12 + rmIL-18, rmIFN-γ, rmM-CSF or rmGM-CSF (–)2 hours before infection with the corresponding LD90 dose of Lm, with or without Amp; control animals received PBS/HSA or PBS/NS in lieu of cytokines, with or without Amp (Tables 4 and 5). Mortality was monitored and recorded daily for 7 days.
Prophylactic Sequential Doses of Cytokines Litters of neonatal mice (n = 10) received sequential doses of cytokines consisting of rmM-CSF at (–)18 hours, rmIL-12 and/ or rmIL-18 at (–)6 hours and rmIFN-γ at (–)2 hours before the administration of the LD90 dose of Lm, with or without Amp. Control animals received PBS/HSA in lieu of cytokines, with or without Amp (Tables 4 and 5).
Statistical Methods Overall survival was estimated for each treatment group using the product-limit method of Kaplan–Meier and was compared between groups using the log-rank test. A P value of
Enhanced Survival During Experimental Listeria monocytogenes Sepsis in Neonatal Mice Prophylactically Treated With Th1 and Macrophage Immunoregulatory Cytokines and Mediators Mark B. Geyer, MD,* Kavita Radhakrishnan, MD,†‡ Carmella Van de Ven, MA,‡ Mandhir S. Suri, MD,§ Janet Ayello, MS,‡ and Mitchell S. Cairo, MD*‡¶‖**†† Background: Impairments in T-cell and macrophage-mediated host defense lead to increased infection-related morbidity and mortality in neonates, partly because of immaturity in T helper (Th)1 function. Listeria monocytogenes (Lm) is an intracellular pathogen disproportionately causing severe disease among neonates and the immunocompromised. Intact macrophage and Th1-mediated immune responses are critical for Lm clearance. We and others have previously demonstrated downregulation of Th1 and macrophage immunoregulatory cytokines in cord blood versus adult peripheral blood. We sought to determine whether therapeutic or prophylactic single agent or combination recombinant murine interleukin (rmIL)12, rmIL-18, recombinant murine macrophage-colony stimulating factor (rmM-CSF), recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) and recombinant murine interferon (rmIFN)-γ would enhance survival during experimental neonatal Lm sepsis. Methods: A 90% lethal dose (LD90) of Lm was established in C57/BL/6 neonatal mice. rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ were administered singly or sequentially, before or after LD90 Lm inoculation; ampicillin was administered 24 hours after inoculation. Results: Therapeutic doses of rmIL-12, rmIL-18, rmM-CSF, rmGM-CSF and rmIFN-γ as single agents and sequential therapy with rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were not associated with increased survival. However, prophylactic single doses of rmIL-12, rmIL-18, rmM-CSF and rmIFN-γ and prophylactic sequential doses of rmM-CSF + (rmIL-12 and/or rmIL-18) + rmIFN-γ in addition to ampicillin were associated with significantly enhanced survival compared with ampicillin alone. Conclusions: These data suggest prophylactic administration of macrophage and Th1 immunoregulatory cytokines can potentially overcome deficits in neonatal immunity to protect against Lm. Key Words: Listeria monocytogenes, neonatal, cytokine, interleukins, host defense (Pediatr Infect Dis J 2014;33:e330–e337)
Accepted for publication May 29, 2014. From the *Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; †Department of Pediatrics, University of California San Francisco, San Francisco, CA; ‡Department of Pediatrics, New York Medical College, Valhalla, NY; §Department of Pediatrics, Children’s Hospital and Health Center, San Diego, CA; and Department of ¶Medicine, ‖Pathology, **Microbiology and Immunology, and ††Cell Biology and Anatomy, New York Medical College, Valhalla, NY. Presented in part at the Pediatric Academic Societies Meeting, May 2003, Seattle, WA, and the American Society of Pediatric Hematology/Oncology Meeting, April–May 2004, San Francisco, CA. New York Medical College (PI: M.S.C.) received a grant from the Pediatric Cancer Research Foundation (Irvine, CA) in part to support this work. The authors have no other funding or conflict of interest to disclose. Address for correspondence: Mitchell S. Cairo, MD, Departments of Pediatrics, Medicine, Pathology, Microbiology and Immunology, and Cell Biology and Anatomy, New York Medical College, 19 Skyline Drive, Room IN-D12, Valhalla, NY 10595. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 0891-3668/14/3312-e330 DOI: 10.1097/INF.0000000000000442
e330 | www.pidj.com
L
isteria monocytogenes (Lm), a small, facultative anaerobe, gram-positive bacillus, can cause severe foodborne illness and remains a major cause of life-threatening bacterial infection in neonates. It is estimated that 2500 cases of severe listeriosis occur in the United States annually, mostly among pregnant women, neonates and immunocompromised adults, causing some 2300 hospitalizations and 500 deaths.1,2 Nonperinatal listeriosis-related deaths have decreased in the past 2 decades, presumably secondary to closer monitoring of food manufacturing.3 However, despite the widespread availability of antibiotic treatments for listeriosis, reported case fatality rates for neonates have ranged from 40% to 56%.4 Neonatal listeriosis may take the form of bacteremia and/or CNS infections. Early-onset Listeria sepsis is generally associated with maternal infection, premature delivery and a high case fatality rate. Lm is readily phagocytosed by host macrophages and other nucleated cells and incorporated into a phagolysosome. Lm subsequently lyses the phagolysosome through the actions of a secreted bacterial hemolysin (listeriolysin O) and enters the cytosol, where it directs polymerization of host-cell actin through the ActA transmembrane protein to achieve motility within the cytosol and spread from cell to cell. Because Lm is an intracellular pathogen, intact macrophage and cell-mediated immunity are critical to successful host defense.5 In adult mice, increased production of interferon (IFN)-γ by Lm-infected mice leads to macrophage activation and clearance of Lm infection.6,7 Colony stimulating factor (CSF)-1 and macrophage (M)-CSF regulate differentiation, proliferation and survival of macrophages and serve as a macrophage chemoattractants, and interleukin (IL)-12 and IL-18 act synergistically to induce IFN-γ production by natural killer (NK) and T helper (Th)1 cells during the host immune response to Lm infection. Neonates are characterized by their increased susceptibility to infection, particularly when T-cell and/or macrophage-mediated immune responses are required, by reduced responses to vaccineinduced antigenic stimulation and by the highest rates of infectious mortality in the human lifespan.8 In part, intrinsic bias against Th1 immune responses in the neonate has been implicated in increased susceptibility to bacterial infection.9,10 IFN-γ protein production and gene expression from stimulated umbilical cord blood (UCB) versus adult peripheral blood (PB) mononuclear cells (MNC) are significantly decreased.11–13 We have previously demonstrated significant downregulation of IL-12, IL-18, M-CSF and granulocyte macrophage (GM)-CSF mRNA production and decreased posttranscriptional stability in stimulated UCB versus adult PB MNC.14–17 Given the deficits observed in Th1 and macrophage immunoregulatory cytokines in neonatal cord blood and the mortality observed in neonates despite prompt antibiotic therapy in Lm sepsis and the observed roles of IL-12, IL-18 and M-CSF in the adult murine host response to Lm infection, we hypothesized that prophylactic administration of these cytokines might have a protective effect in Lm sepsis in a neonatal mouse model. In this study, we therefore assessed the effects on survival of exogenous administration of prophylactic doses of Th1 and macrophage
The Pediatric Infectious Disease Journal • Volume 33, Number 12, December 2014
The Pediatric Infectious Disease Journal • Volume 33, Number 12, December 2014
immunoregulatory cytokines and mediators, singly and sequentially, including IL-12, IL-18, M-CSF and GM-CSF in neonatal mice during experimental Lm sepsis.
MATERIALS AND METHODS Animals Adult wild-type (WT) C57BL/6 mice (20–25 g) and litters of C57BL/6 WT neonatal mice (≤24 hours old; 1.2–1.7 g) were used for this study. All animals were obtained from Jackson Laboratories, Bar Harbor, ME. Mothers of C57BL/6 litters were received 1 week before delivery. All animals were housed in the vivarium at Columbia University and maintained at constant room temperature with water and rodent feed ad libitum. Neonatal mice were pooled and then randomly split as equally as possible to each of the mothers; this was done in order to avoid any litter bias in the experiments. Approval for this study was obtained from the Institutional Animal Care and Use Committee at Columbia University.
Organism Lm (ATCC 7644, Manassas, VA) was grown in brain heart infusion to logarithmic phase, aliquoted, and stored at –70°C until use. Bacterial suspensions of 3 × 105 – 3 × 108 organism/g body weight/100 μL were prepared in phosphate buffered saline (PBS)/ human serum albumin (HSA) for 90% lethal dose (LD90) and cytokine studies.
Cytokines Recombinant murine (rm) IL-12 (0.01 μg/g body weight), rmIL-18 (0.1 μg/g body weight), rmIFN-γ (5 μg/pup), rmM-CSF (5 μg/kg body weight) and rmGM-CSF (3 μg/kg body weight) (R & D Systems Inc, Minneapolis, MN) were used in the study. All cytokines were purchased from R&D Systems, Inc. (Minneapolis, MN). Endotoxin levels in each batch were confirmed to be 10%) were euthanized and considered nonsurvivors.
Antibiotic Ampicillin (Amp) 100 mg/kg subcutaneous q12h was administered 24 hours after Lm injection to specific subgroups of Lm-infected animals.
Therapeutic Single or Repeated Dose of Cytokines Litters of neonatal mice (n = 10–15) received a dose of rmIL-12, rmIL-18, rmIL-12 + rmIL-18, rmIFN-γ, rmM-CSF or rmGM-CSF at 6 hours (single dose) or 6 hours and 24 hours (repeated dose) after infection with the LD90 dose of Lm, with or without Amp. Control animals received PBS/normal saline (NS) in lieu of cytokines, with or without Amp (Tables 1–3). Mortality was monitored and recorded daily for up to 7 days. © 2014 Lippincott Williams & Wilkins
Cytokine Prophylaxis in Listeria Sepsis
Therapeutic Sequential Doses of Cytokines Litters of neonatal mice (n = 10) received sequential doses of cytokines consisting of rmM-CSF at 2 hours, rmIL-12 and/or rmIL-18 at 6 hours and rmIFN-γ at 18 hours after the administration of the LD90 dose of Lm with or without Amp. Control animals received PBS/NS in lieu of cytokines with or without Amp (Tables 1–3). Mortality was monitored and recorded daily for 7 days.
Prophylactic Single Dose of Cytokines Litters of neonatal mice (n = 10–12) received a single dose of rmIL-12, rmIL-18, rmIL-12 + rmIL-18, rmIFN-γ, rmM-CSF or rmGM-CSF (–)2 hours before infection with the corresponding LD90 dose of Lm, with or without Amp; control animals received PBS/HSA or PBS/NS in lieu of cytokines, with or without Amp (Tables 4 and 5). Mortality was monitored and recorded daily for 7 days.
Prophylactic Sequential Doses of Cytokines Litters of neonatal mice (n = 10) received sequential doses of cytokines consisting of rmM-CSF at (–)18 hours, rmIL-12 and/ or rmIL-18 at (–)6 hours and rmIFN-γ at (–)2 hours before the administration of the LD90 dose of Lm, with or without Amp. Control animals received PBS/HSA in lieu of cytokines, with or without Amp (Tables 4 and 5).
Statistical Methods Overall survival was estimated for each treatment group using the product-limit method of Kaplan–Meier and was compared between groups using the log-rank test. A P value of
