Involvement of type I immune responses in swine-origin H1N1 influenza virus infection.
ABSTRACT Swine-origin H1N1 influenza virus (S-OIV) appeared in 2009 with a higher incidence rate among children. Although fever was the most common symptom, some complicated cases occurred. We evaluated the percentages of effector T cells, B cells, and regulatory T cells in peripheral blood from 5 children infected by S-OIV (1 with acute necrotizing encephalitis, 2 with pneumonia, and 2 without complications), 5 children with seasonal influenza, and 5 healthy children. We found higher percentages of T-bet(+) CD4(+)CD8(+) T cells, monocytes, and B cells, granzyme B(+) and perforin(+) CD4(+), and CD8(+) T cells in affected children with both seasonal and H1N1 influenza than in controls, whereas both groups demonstrated similar percentages of CD4(+)CD25(+)Foxp3(+) regulatory T cells. In infected children with complications we observed high percentages of perforin(+) and interferon-γ(+) CD4(+) and CD8(+) T cells associated with low percentages of T regulatory cells. Our data suggest a dysregulation of antipathogen type I immune responses in complicated S-OIV infections.
- SourceAvailable from: Stephen Mutoloki[show abstract] [hide abstract]
ABSTRACT: Acquired immunity plays an important role in the protection of salmonids vaccinated against infectious pancreatic necrosis virus (IPNV) infections. In recent years, vaccine research has taken a functional approach to find the correlates of protective immunity against IPNV infections. Accumulating evidence suggests that the humoral response, specifically IgM is a correlate of vaccine protection against IPNV infections. The role of IgT on the other hand, especially at the sites of virus entry into the host is yet to be established. The kinetics of CD4+ and CD8+ T-cell gene expression have also been shown to correlate with protection in salmonids, suggesting that other arms of the adaptive immune response e.g. cytotoxic T cell responses and Th1 may also be important. Overall, the mechanisms of vaccine protection observed in salmonids are comparable to those seen in other vertebrates suggesting that the immunological basis of vaccine protection has been conserved across vertebrate taxa.Developmental and comparative immunology 08/2013; · 3.29 Impact Factor
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ABSTRACT: Objective: Obese adults have a greater risk of morbidity and mortality from infection with pandemic H1N1 influenza A virus (pH1N1). The objective of the present study was to elucidate the specific mechanisms by which obesity and overweight impact the cellular immune response to pH1N1.Design and Methods: We stimulated peripheral blood mononuclear cells from healthy weight, overweight, and obese individuals ex vivo with live pH1N1 and then measured markers of activation and function using flow cytometry and cytokine secretion using cytometric bead array assays.Results: Our data indicate that CD4(+) and CD8(+) T cells from overweight and obese individuals expressed lower levels of CD69, CD28, CD40 ligand, and interleukin-12 receptor, as well as, produced lower levels of interferon-γ and granzyme B, compared to healthy weight individuals, suggesting deficiencies in activation and function. Dendritic cells from the three groups expressed similar levels of major histocompatibility complex-II, CD40, CD80, and CD86, as well as, produced similar levels of interleukin-12.Conclusions: The defects in CD4(+) and CD8(+) T cells may contribute to the increased morbidity and mortality from pH1N1 in obese individuals. These data also provide evidence that both overweight and obesity cause impairments in immune function.Obesity 03/2013; · 3.92 Impact Factor
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ABSTRACT: Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction.Virology 01/2014; 448:91–103. · 3.35 Impact Factor
Involvement of type I immune responses in swine-origin H1N1 influenza
Giovanni Frisulloa, Raffaele Iorioa, Domenico Plantonea, Viviana Nocitia,b, Agata Katia Patanellaa,b,
Alessandro Martia, Concetta Palermoc, Piero Valentinid, Paolo Mariottic, Anna Paola Batocchia,*
aInstitute of Neurology, Department of Neurosciences, Catholic University, Rome, Italy
bDon Carlo Gnocchi Foundation, Milan, Italy
cChild Neuropsychiatry Unit, Catholic University, Rome, Italy
dDepartment of Paediatric Sciences, Catholic University, Rome, Italy
A R T I C L EI N F O
Received 10 June 2010
Accepted 18 April 2011
Available online 3 May 2011
Swine-origin H1N1 influenza virus
A B S T R A C T
Swine-origin H1N1 influenza virus (S-OIV) appeared in 2009 with a higher incidence rate among children.
Although fever was the most common symptom, some complicated cases occurred. We evaluated the
percentages of effector T cells, B cells, and regulatory T cells in peripheral blood from 5 children infected by
S-OIV (1 with acute necrotizing encephalitis, 2 with pneumonia, and 2 without complications), 5 children
with seasonal influenza, and 5 healthy children. We found higher percentages of T-bet?CD4?CD8?T cells,
monocytes, and B cells, granzyme B?and perforin?CD4?, and CD8?T cells in affected children with both
seasonal and H1N1 influenza than in controls, whereas both groups demonstrated similar percentages of
of perforin?and interferon-??CD4?and CD8?T cells associated with low percentages of T regulatory cells.
Our data suggest a dysregulation of antipathogen type I immune responses in complicated S-OIV infections.
? 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
In early 2009, a novel pandemic, swine-origin H1N1 influenza
virus (S-OIV), appeared, but it has not exhibited unusually high
pathogenicity . S-OIV infection exhibited a higher incidence
rate among children but the majority of cases have been mild .
However, severe illnesses and death mostly occurred in children
younger than 5 years of age and in children with high-risk
conditions . The clinical presentation of S-OIV infection is
largely indistinguishable from that of seasonal influenza symp-
toms; it is characterized by cough, headache, rhinorrhea, ab-
sence of leukocytosis, and a normal chest radiograph . The
main complication is extension of viral infection to the alveoli,
often associated with secondary bacterial infection, resulting
in severe pneumonia . Neurological complications occur
The host immune response seems to play a role in the evolu-
tion of influenza virus infection. A recent study demonstrated an
activation of innate immunity with high serum levels of che-
moattractants of monocytes and macrophages such as IP-10,
MIG, and MCP-1 in all S-OIV patients, whereas an activation of
adaptive immunity with increased expression of T-helper
(Th)-1- and Th17-correlated cytokines was observed only in
severe cases .
T-bet has been identified as a key transcription factor for the
development of interferon-? (IFN-?)-producing Th1 cells  and
the induction of lytic granules such as perforin and granzyme in
cytotoxic CD8?T cells .
Infection with more pathogenic strains of influenza requires
both B and T cells for complete clearance. In addition to antibody-
producing B cells, both Th1 cells and cytotoxic CD8?T cells seem to
be involved in the protective immune response to influenza virus
infection , whereas the role of Th17 cells is still controversial
[9,10]. Regulatory T cells (Treg) suppress host immune responses
against self- or nonself-antigens, thus playing a critical role in the
prevention of autoimmune diseases and in the modulation of im-
mune responses to pathogens . Foxp3 is a transcription factor
and function .
To assess the relevance of host immune response in the evolu-
tion of S-OIV infection, we evaluated the percentages of CD4?,
CD8?T cells, CD14?monocytes, and CD19?B cells positive for
T-bet; the percentages of CD4?, CD8?T cells positive for perforin
and granzyme B; the percentage of CD4?CD25?Foxp3?regulatory
T cells, and the mean Foxp3 expression in CD4?T cells using flow
cytometry in peripheral blood from 5 children affected by compli-
cated and uncomplicated S-OIV infection and 5 age- and sex-
* Corresponding author.
E-mail address: firstname.lastname@example.org (A.P. Batocchi).
Human Immunology 72 (2011) 632-635
Contents lists available at ScienceDirect
0198-8859/11/$32.00 - see front matter ? 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
matched controls. In addition, we evaluated the percentage of
CD4?and CD8?T cells positive for IFN-? and interleukin (IL)-17 by
2. Subjects and methods
Five previously healthy hospitalized children with S-OIV infec-
tion confirmed by molecular diagnostic methods, 5 children with
seasonal influenza without signs of bacterial or other viral infec-
tions in respiratory, urinary, and blood cultures, and 5 age- and
sex-matched healthy controls were included in the study.
No child had been previously vaccinated for seasonal influenza.
No child had taken antiviral or antibiotic therapy before blood
sampling. All blood samples were obtained within 4 days from the
onset of the first symptoms. Informed consent was obtained from
parents of children before enrollment and the study was approved
by the Catholic University ethics committee.
2.2. Viral diagnosis
Influenza virus was detected by immunofluorescence assay and
confirmed by reverse transcriptase polymerase chain reaction for
detected on nasal swabs using a TaqMan assay.
2.3. Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated from
venous blood by density gradient centrifugation (2500g, 30 min-
utes) over a Ficoll–Hypaque density gradient (Pharmacia, Uppsala,
Sweden). PBMC were then harvested by pipetting cells from the
Ficoll/serum interface and washed twice.
2.4. Flow cytometry
Isolated PBMC were washed once in culture medium (RPMI me-
dium) containing fetal calf serum and once in phosphate-buffered
saline (PBS) and incubated with the specific phycoerythrin–
cyanine 5 (PC5)-conjugated antibody (CD4, CD8, CD14, CD19;
Beckman Coulter, Miami, FL). PBMC were then fixed with 2% para-
formaldehyde for 10 minutes. Detection of T-bet, perforin, and
granzyme was performed by intracellular flow cytometry using
nology, Santa Cruz, CA), anti perforin–FITC antibody (clone dG9e,
eBioscience, San Diego, CA), and anti granzyme–PE antibody (clone
GB11, eBioscience). After fixation, cells were washed once. Cells
were then permeabilized using a commercially available perm/
wash kit (eBioscience). Upon permeabilization, 3 ? 105cells were
PBS and resuspended in PBS for flow cytometry (Beckman Coulter,
EPICS XL). Each analysis was performed using at least 50,000 cells,
which were gated in the region of the lymphocyte–monocyte pop-
ulation, as determined by light scatter properties (forward scatter
vs side scatter). To analyze the expression of T-bet in monocytes,
cells were gated in both the monocyte (morphologic gate) and the
CD14?(immunologic gate) regions. To assess the expression of
transcription factors in lymphocytes (CD4?/CD8?/CD19?cells),
cells were gated in both the lymphocyte and the CD4?/CD8?/
CD19?regions. Appropriate fluorochrome-conjugated isotype-
matched monoclonal antibodies were used as control for back-
ground staining in each flow acquisition.
For the detection of Foxp3 expression, PBMC were analyzed by
three-color intracellular flow cytometry using anti-CD4–PC5 con-
jugate (Beckman Coulter), anti-CD25–FITC conjugate (Beckman
236A/E7; eBioscience). In particular, isolated PBMC were washed
once in culture medium (Dulbecco’s modified Eagle’s medium)
anti-CD4–PC5 and anti-CD25–FITC. After fixation, cells were per-
meabilized using a commercially available permeabilization/wash
kit (eBioscience). Upon permeabilization, 5 ? 105cells were resus-
pended in PBS and incubated for 30 minutes with the anti-
Foxp3–PE conjugate. Cells were washed again with cold PBS and
resuspended in PBS for flow cytometry (EPICS XLTM; Beckman
2.5. Cytokine intracellular staining
For intracellular staining, cells were stimulated for 5 hours with
mycin (1 ?g/mL; Sigma) in the presence of brefeldin A (9 ?g/mL;
eBioscience). Cells were first stained with anti-CD4– and anti-
CD8–PC5-conjugated antibodies (Beckman Coulter) and then fixed
and made permeable (Fix/Perm; eBioscience) according to the
manufacturer’s instructions. Intracellular cytokine staining was
performed using PE-conjugated antibodies specific for human
IFN-? and IL-17 (eBioscience). Appropriate fluorochrome-matched
staining. Flow cytometric analysis was conducted by gating on
lymphocyte and CD4?and CD8?cells.
2.6. Statistical analysis
Differences among patients with H1N1 and seasonal influenza
and controls in percentages of CD4?and CD8?T cells, CD14?
monocytes, and CD19?B cells expressing transcription factors and
in the percentages of perforin?and granzyme B?CD4?and CD8?T
cells were assessed using the Mann–Whitney U test. Statistical
significance was set at p ? 0.05.
males and 3 females) with ages ranging from 2 to 12 years, 5
children with seasonal influenza (3 males and 2 females) with ages
ranging from 3 to 14 years, and 5 age- and sex-matched healthy
children. Among children with S-OIV infections, 2 children exhib-
ited no complications and 2 had pneumonia. One child exhibited
acute necrotizing encephalitis (Pt. 5) and required admission to an
intensive care unit because of her neurological condition. All chil-
sequelae (divergent strabismus of the left eye and a gait distur-
bance) upon hospital discharge. In the seasonal cohort no compli-
cation was observed. Demographic and clinical features of the
patients are summarized in Table 1.
and in children with seasonal influenza than in controls (p ? 0.011,
0.009, 0.006, and 0.009 and p ? 0.026, 0.036, 0.011, and 0.024,
respectively; Fig. 1A). Percentages of granzyme B?and perforin?
CD4?and CD8?T cells were increased both in S-OIV (p ? 0.016 and
percentages of CD4?perforin?T cells and CD8?perforin?T cells
were significantly higher in children with S-OIV infection than in
percentage of CD4?CD25?Foxp3?T cells among affected children
ages of T-bet?, perforin?, and granzyme B?CD4?and CD8?T cells
but a similar percentage of T-bet?CD14?monocytes and B cells
(Fig. 1A). In addition, complicated cases exhibited low percentages
of CD4?CD25?Foxp3?T cells expressing low levels of Foxp3. The
percentages of IFN-??CD4?and CD8?T cells were higher in the
peripheral blood both from S-OIV infected children (p ? 0.009 and
G. Frisullo et al. / Human Immunology 72 (2011) 632-635
0.008, respectively; Fig. 1A) and from children with seasonal influ-
Moreover, in peripheral blood of S-OIV-infected children we ob-
served significantly higher percentages of both CD4?IFN-??and
CD8?IFN-??T cells than in children with seasonal influenza (p ?
0.042 and 0.018, respectively; Fig. 1A). We demonstrated very low
percentages of IL-17?CD4?and CD8?T cells both in patients and
in controls. Patient 5 with acute necrotizing encephalitis exhib-
ited the highest percentages of CD8?T cells positive for T-bet,
IFN-?, granzyme B, and perforin and the lowest percentage of
CD4?CD25?Foxp3?T cells that expressed the lowest levels of
Foxp3 (Fig. 1B). Representative 2-parameter plots of T-bet?,
Demographic and clinical features of S-OIV and seasonal influenza A/B–infected children
Patients Age (years)SexOnset symptomsComplications Antiviral therapiesOutcome
Fever, vomiting, cough
Oseltamivir, 90 mg a day for 5 days
Oseltamivir, 60 mg a day for 5 days
Oseltamivir, 60 mg a day for 5 days
Oseltamivir, 150 mg a day for 5 days
Oseltamivir, 60 mg a day, methylprednisolone,
1 mg/kg/day for 5 days
6 (influenza A)
7 (influenza A)
8 (influenza B)
9 (influenza B)
10 (influenza A)
Fig. 1. (A) Percentage of CD4?T-bet?and CD8?T-bet?T cells, CD14?T-bet?and CD19?T-bet?B cells, CD4?and CD8?granzyme?T cells, CD4?and CD8?perforin?T cells,
CD4?CD25?Foxp3?T cells, CD4?and CD8?IFN-??T cells, and CD4?and CD8?IL-17?T cells from peripheral blood of 5 children with swine-origin H1N1 influenza virus
(S-OIV), 5 children with seasonal influenza infection, and 5 age-matched healthy controls. (B) Representative 2-parameter plots indicating cells gated on CD4?CD8?T cells,
CD14?monocytes, and CD19?B cells from a child with acute necrotizing encephalitis by S-OIV (Pt. 5), a child with confirmed S-OIV infection without complications (Pt. 2),
and a control (Ctrl. 3). The y axis of each histogram represents the specific fluorescence of T-bet–PE and perforin–FITC; the x axis represents the specific fluorescence of
extracellular CD4–, CD8–, CD14–, and CD19–PE–Cy5 on 4 decade logarithmic scales. These representative 2-parameter plots are obtained from the same patient during
relapse and remission. Quadrants were set using appropriate isotype controls for each intra- and extracellular antibody.
G. Frisullo et al. / Human Immunology 72 (2011) 632-635
perforin?T, B cells, and monocytes from Pts. 5 and 2 and control
3 are presented in Fig. 1B.
The balance between effector immune and Treg functions can
influence the outcome of host–microorganism coexistence. During
viral infection, the activation of the innate immune system is es-
sential for subsequent adaptive immune responses, including spe-
cific antibody production, and plays a crucial key role in protection
against virus infection.
In our study we reported a high percentage of circulating
activation of innate immunity is always present both in S-OIV
infection and in seasonal influenza as demonstrated by other au-
thors . Moreover, our data agree with the results of another
study  in which the authors demonstrate that the pandemic
H1N1 virus induces a similar cytokine-mediated immune response
in human dendritic cells and macrophages as the seasonal influ-
enza viruses. However, we also observed a pronounced activation
of T lymphocytes as demonstrated by the higher percentages of
circulating CD4?and CD8?T cells producing perforin and IFN-? in
S-OIV-infected children than in children with seasonal influenza.
These data agree with the results of Ge and colleagues , who
demonstrated that H1N1 viruses led to the generation of specific
memory T cells.
All affected children exhibited increased percentages of
CD19?T-bet?B cells, indicative of a dominant Be1 response 
Similar findings have been reported in mice in which the immuno-
globulin G (IgG) subclass responses to influenza are dominated by
of T-bet in primary B cells . The percentages of T-bet?, IFN-??,
perforin?, and granzyme B?CD4?and CD8?T cells were higher in
children with S-OIV and seasonal influenza infection than in con-
trols and complicated S-OIV patients exhibited high percentages of
these T cell subsets. Our data agree with the autopsy findings of
patients with confirmed S-OIV infection and acute respiratory fail-
ure that indicated necrotizing bronchiolitis with marked expres-
sion of IFN-? and a large number of CD8?T cells and granzyme B?
cells within the lung tissue .
We demonstrated higher percentages of IFN-??CD4?and CD8?
T cells in affected children than in controls, whereas low percent-
ages of IL-17?CD4?and CD8?T cells were present both in patients
We did not report any difference in the percentages of
CD4?CD25?Foxp3?T cells and in the levels of Foxp3 expression in
CD4?CD25?T cells between patients and controls. In the S-OIV-
T cells expressing T-bet, perforin, and granzyme, suggesting a role
for regulatory cells in the regulation of the host immune response.
Our data suggest that both S-OIV and seasonal influenza infec-
tion induce an activation of innate and adaptive immunity with a
prominent polarization toward a type 1 immune response. How-
ever, S-OIV infection, compared with seasonal influenza, seems to
ing IFN-? and perforin, which can determine a local and systemic
overresponse of the immune system and, consequently, influenza
Because the limitations of this study include its modest sample
size, further studies in more extensive series of patients are neces-
sary to better understand the role of host immune response in the
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