Type 1 Interferon Induction of Natural Killer Cell Gamma Interferon
Production for Defense during Lymphocytic Choriomeningitis Virus
Ethan A. Mack, Lara E. Kallal, Delia A. Demers, and Christine A. Biron
Division of Biology and Medicine, Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA
Received 19 July 2011 Accepted 21 July 2011 Published 9 August 2011
Citation Mack EA, Kallal LE, Demers DA, Biron CA. 2011. Type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic
choriomeningitis virus infection. mBio 2(4):e00169-11. doi:10.1128/mBio.00169-11.
Editor Diane Griffin, Johns Hopkins University School of Public Health
Copyright © 2011 Mack et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported
License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to Christine A. Biron, Christine_Biron@brown.edu.
as a result of their cytotoxicity and cytokine-producing abilities,
effects are incompletely understood. During responses to viral
infections, the antiviral cytokines, type 1 interferons (IFN-?/?)
stimulate both cellular resistance to viruses and NK cell cytotoxic
function (3–5). The cytokines also have the potential to either
but type 1 IFN enhancement of IFN-? might not be important in
NK cell responses to viruses because infections eliciting high sys-
temic type 1 IFN levels are not associated with systemic NK cell
is induced and is dependent on this cytokine (4, 8). As a conse-
and immunoregulatory functions (1, 2). They mediate these
tions with viruses failing to stimulate IL-12 production.
The first described signaling pathway engaged by type 1 IFN
binding to the specific heterodimeric receptor stimulates phos-
phorylation of the signaling and transcription factors STAT1 and
STAT2 (5, 10). Complexes, including these activated intermedi-
IFN immunoregulatory effects, including activation of NK cell
cytotoxicity, are dependent on STAT1 (4, 11). There are a total of
seven STAT molecules—STAT1 through STAT6, with two
STAT5s—and type 1 IFNs conditionally activate all of these (5,
12), including STAT4, an important intermediary in IL-12 stim-
ulation of NK cells as well as type 1 IFN stimulation of certain T
cell populations for IFN-? production (4, 13–15). Previous work
July/August 2011 Volume 2 Issue 4 e00169-11
has only been possible to identify the type 1 IFN induction of NK
cell IFN-? production during acute viral infections of STAT1-
induction of STAT1 by type 1 IFN and/or IFN-? negatively regu-
lates the response (6, 9). These results leave open the intriguing
question of why a pathway from type 1 IFN to STAT4 activation
when it is rapidly turned off at times of type 1 IFN production.
earliest times of viral infection under immunocompetent condi-
tions. The system used for study was intraperitoneal (i.p.) infec-
tion of C57BL/6 (B6) mice with lymphocytic choriomeningitis
virus (LCMV) (7, 9, 16, 17). This infection has been well charac-
can cause significant morbidity and mortality, particularly in im-
munodeficient individuals (18–20). The virus is a potent inducer
of type 1 IFN, with systemic levels produced in serum and spleen
for several days after i.p. infection (7, 9, 17). In contrast, LCMV is
a poor inducer of IL-12, with low to undetectable levels of the
cytokine produced (8, 16), and NK cell IFN-? expression is also
low to undetectable in the serum and spleens of LCMV-infected
mice (8, 9). Because the initial site of virus entry is the peritoneal
The results of the experiments reported here reveal unique
production and NK cell IFN-? expression detected at 24 h after
infection and peaking at 30 h after infection. Studies under con-
ditions of blocked type 1 IFN receptor access and/or deficiency in
STAT4 conclusively demonstrated that NK cell IFN-? expression
was dependent on the type 1 IFNs and STAT4. The pathway en-
hanced the antiviral state. Interestingly, in comparison to other
peritoneal populations, STAT1 induction in NK cells was delayed
and separated from the NK cell IFN-? response by 10 to 12 h.
cell IFN-? and mechanistically resolve important issues concern-
ing the earliest pathways to, sources of, and functions for IFN-?.
Innate cytokine responses to LCMV infection in the peritoneal
acterize responses at the earliest times after infections, lavage flu-
had been uninfected or infected i.p. with 5 ? 104PFU LCMV at
tion (Fig. 1A). Enzyme-linked immunosorbent assays (ELISAs)
were used to measure IFN-? and IFN-?. In contrast to the re-
in serum samples taken from mice used for these experiments
toneum reached their peak values of 340 and 69 pg/ml, respec-
tively, at 30 h after challenge and were sharply confined to the 6-h
known inducer of IFN-?, were acquired from the CBAs. All sam-
ples were below the limit of detection for the assay, with no de-
tectable levels in a large majority of samples isolated after 20 to
to LCMV infection include early induction of IFN-?, with kinet-
To define the major cell populations contributing to IFN-?
production, peritoneal excudate cells (PECs) from uninfected
48 h were isolated, stained for NK1.1 and T cell receptor ? (TCR-
?), fixed, and permeabilized to stain for cytoplasmic IFN-? (see
Materials and Methods for staining and gating strategies). Total
lymphocytes were evaluated with an extended lymphocyte gate.
The NK cell subpopulations were identified as NK1.1?TCR-??
and the T cell subpopulations as NK1.1?TCR-??. Lymphocytes
isolated from uninfected mice, including total, NK, and T cells,
did not basally express detectable IFN-?; the positive frequencies
for all populations were ?4% (Fig. 1B and C). By 24 h after chal-
lenge with LCMV, approximately 14 to 37% of the NK cells ex-
this NK cell response was at 30 h after infections, when 28 to 43%
of the NK cells expressed IFN-?. The response was resolving at 36
cell populations had high IFN-? levels at any of the times exam-
ined (Fig. 1B and C). These results demonstrate that during
LCMV infection, peritoneal NK cells respond with IFN-? expres-
Type 1 IFN responsiveness is required for induction of NK
cell IFN-? expression. Because induction of the biologically ac-
tive IL-12p70 heterodimer can be accompanied by higher-level
IL-12p40 subunit production (8), representative lavage samples
from 0, 24, 30, 36, 40, and 48 h following LCMV infection were
tested in an ELISA for the subunit. Consistent with our earlier
detection for the assay (data not shown). To determine if biolog-
ically relevant IL-12 was induced below detection limits, unin-
fected and LCMV-infected mice were treated with control anti-
body or anti-IL-12p40 antibody neutralizing the function of
ride (LPS) i.p. at 6 h prior to harvest. In comparison to LCMV
infection, LPS treatment induced higher levels of IFN-? in lavage
fluids (Fig. 2A) and higher proportions of NK cells expressing
anti-IL-12 treatment. In contrast, the IFN-? response to LCMV
by the virus.
1 IFNs in stimulating NK cell IFN-? within mice blocked in their
responsiveness to the cytokines. The first examined responses in
cells from uninfected and 30-h LCMV-infected immunocompe-
tent mice that had been treated with control antibodies or anti-
bodies blocking access to the type 1 IFN receptor, anti-IFNAR
(23). As shown in Fig. 3A, the infected control-treated mice had
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9. Miyagi T, et al. 2007. High basal STAT4 balanced by STAT1 induction to
control type 1 interferon effects in natural killer cells. J. Exp. Med. 204:
10. Platanias LC. 2005. Mechanisms of type-I- and type-II-interferon-
mediated signalling. Nat. Rev. Immunol. 5:375–386.
11. Lee CK, et al. 2000. Distinct requirements for IFNs and STAT1 in NK cell
function. J. Immunol. 165:3571–3577.
12. Brierley MM, Fish EN. 2002. Review: IFN-alpha/beta receptor interac-
tions to biologic outcomes: understanding the circuitry. J. Interferon Cy-
tokine Res. 22:835–845.
13. Kaplan MH, Sun YL, Hoey T, Grusby MJ. 1996. Impaired IL-12 re-
sponses and enhanced development of Th2 cells in Stat4-deficient mice.
14. Rogge L, et al. 1998. The role of Stat4 in species-specific regulation of Th
cell development by type I IFNs. J. Immunol. 161:6567–6574.
15. Nguyen KB, et al. 2002. Critical role for STAT4 activation by type 1
interferons in the interferon-gamma response to viral infection. Science
16. Cousens LP, et al. 1999. Two roads diverged: interferon alpha/beta- and
interleukin 12-mediated pathways in promoting T cell interferon gamma
responses during viral infection. J. Exp. Med. 189:1315–1328.
17. Louten J, van Rooijen N, Biron CA. 2006. Type 1 IFN deficiency in the
gitis virus infection. J. Immunol. 177:3266–3272.
18. Barton LL, Mets MB. 2001. Congenital lymphocytic choriomeningitis
virus infection: decade of rediscovery. Clin. Infect. Dis. 33:370–374.
19. Fischer SA, et al. 2006. Transmission of lymphocytic choriomeningitis
virus by organ transplantation. N. Engl. J. Med. 354:2235–2249.
20. Palacios G, et al. 2008. A new arenavirus in a cluster of fatal transplant-
associated diseases. N. Engl. J. Med. 358:991–998.
21. Wysocka M, et al. 1995. Interleukin-12 is required for interferon-gamma
production and lethality in lipopolysaccharide-induced shock in mice.
Eur. J. Immunol. 25:672–676.
22. Nguyen KB, Biron CA. 1999. Synergism for cytokine-mediated disease
during concurrent endotoxin and viral challenges: roles for NK and T cell
IFN-gamma production. J. Immunol. 162:5238–5246.
23. Sheehan KC, et al. 2006. Blocking monoclonal antibodies specific for
mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immu-
nized by in vivo hydrodynamic transfection. J. Interferon Cytokine Res.
24. Müller U, et al. 1994. Functional role of type I and type II interferons in
antiviral defense. Science 264:1918–1921.
25. Huang S, et al. 1993. Immune response in mice that lack the interferon-
gamma receptor. Science 259:1742–1745.
26. Wong LH, et al. 2002. Isolation and characterization of a human STAT1
gene regulatory element. Inducibility by interferon (IFN) types I and II
and role of IFN regulatory factor-1. J. Biol. Chem. 277:19408–19417.
27. van Boxel-Dezaire AH, et al. 2010. Major differences in the responses of
primary human leukocyte subsets to IFN-beta. J. Immunol. 185:
28. Zhou H, Zhao J, Perlman S. 2010. Autocrine interferon priming in
macrophages but not dendritic cells results in enhanced cytokine and
29. Miyagi T, et al. 2010. Altered interferon-alpha-signaling in natural killer
cells from patients with chronic hepatitis C virus infection. J. Hepatol.
30. Martinez J, Huang X, Yang Y. 2008. Direct action of type I IFN on NK
cells is required for their activation in response to vaccinia viral infection
in vivo. J. Immunol. 180:1592–1597.
31. Malmgaard L, Paludan SR. 2003. Interferon (IFN)-alpha/beta, interleu-
kin (IL)-12 and IL-18 coordinately induce production of IFN-gamma
during infection with herpes simplex virus type 2. J. Gen. Virol. 84:
response to mature and induce CD4?Th1 immunity with poly IC as
adjuvant. J. Exp. Med. 206:1589–1602.
33. McCartney S, et al. 2009. Distinct and complementary functions of
MDA5 and TLR3 in poly(I:C)-mediated activation of mouse NK cells. J.
Exp. Med. 206:2967–2976.
34. Berenson LS, Farrar JD, Murphy TL, Murphy KM. 2004. Frontline:
absence of functional STAT4 activation despite detectable tyrosine phos-
phorylation induced by murine IFN-alpha. Eur. J. Immunol. 34:
35. Micallef MJ, et al. 1996. Interferon-gamma-inducing factor enhances T
interleukin-12 for interferon-gamma production. Eur. J. Immunol. 26:
36. Sareneva T, Matikainen S, Kurimoto M, Julkunen I. 1998. Influenza A
virus-induced IFN-alpha/beta and IL-18 synergistically enhance IFN-
gamma gene expression in human T cells. J. Immunol. 160:6032–6038.
37. Matikainen S, et al. 2001. IFN-alpha and IL-18 synergistically enhance
IFN-gamma production in human NK cells: differential regulation of
Eur. J. Immunol. 31:2236–2245.
tion of IFN-gamma in response to double-stranded RNA. J. Immunol.
39. Toshchakov V, et al. 2002. TLR4, but not TLR2, mediates IFN-beta-
induced STAT1alpha/beta-dependent gene expression in macrophages.
Nat. Immunol. 3:392–398.
40. Bukowski JF, Woda BA, Habu S, Okumura K, Welsh RM. 1983. Natural
killer cell depletion enhances virus synthesis and virus-induced hepatitis
in vivo. J. Immunol. 131:1531–1538.
41. Bukowski JF, Warner JF, Dennert G, Welsh RM. 1985. Adoptive transfer
studies demonstrating the antiviral effect of natural killer cells in vivo. J.
Exp. Med. 161:40–52.
42. Bukowski JF, Yang H, Welsh RM. 1988. Antiviral effect of lymphokine-
activated killer cells: characterization of effector cells mediating prophy-
laxis. J. Virol. 62:3642–3648.
43. van den Broek MF, Müller U, Huang S, Zinkernagel RM, Aguet M.
1995. Immune defence in mice lacking type I and/or type II interferon
receptors. Immunol. Rev. 148:5–18.
44. Pien GC, Biron CA. 2000. Compartmental differences in NK cell respon-
siveness to IL-12 during lymphocytic choriomeningitis virus infection. J.
45. Biron CA, Sen GC. 2007. Innate immune responses to viral infections, p
Kluwer/Lippincott, Williams & Wilkins, Philadelphia, PA.
46. Miyagi T, Lee SH, Biron CA. 2010. Intracellular staining for analysis of
transcription (STATs) in NK cells. Methods Mol. Biol. 612:159–175.
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