Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza

Abstract

Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1.
We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene.
Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients.
While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

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Vol 13 No 6
Research
Th1 and Th17 hypercytokinemia as early host response signature
in severe pandemic influenza
Jesus F Bermejo-Martin1,2, Raul Ortiz de Lejarazu1,2, Tomas Pumarola3, Jordi Rello4,
Raquel Almansa1,2, Paula Ramírez5, Ignacio Martin-Loeches4, David Varillas1,2, MariaCGallegos
6,
Carlos Serón7, Dariela Micheloud8, Jose Manuel Gomez8, Alberto Tenorio-Abreu9, María J Ramos9,
M Lourdes Molina10, Samantha Huidobro11, Elia Sanchez12, Mónica Gordón5, Victoria Fernández6,
Alberto del Castillo13, Ma Ángeles Marcos3, Beatriz Villanueva14, Carlos Javier López14,
Mario Rodríguez-Domínguez15, Juan-Carlos Galan15, Rafael Cantón15, Aurora Lietor16,
Silvia Rojo1,2, Jose M Eiros1,2, Carmen Hinojosa17, Isabel Gonzalez17, Nuria Torner18,
David Banner19, Alberto Leon20, Pablo Cuesta21, Thomas Rowe19,22 and David J Kelvin19,20,22
1National Centre of Influenza, Hospital Clínico Universitario de Valladolid, Avda Ramón y Cajal 3, Valladolid, 47005, Spain
2Unidad de Investigación en Infección e Inmunidad- Microbiology Service,. Hospital Clínico Universitario de Valladolid- IECSCYL, Avda Ramón y Cajal
3, Valladolid, 47005, Spain
3Virology Laboratory, Hospital Clinic de Barcelona, Carrer de Casanova 143, Barcelona, 08036, Spain
4Critical Care Department, Joan XXIII University Hospital-CIBERes Enfermedades Respiratorias-IISPV. Mallafre Guasch 4, Tarragona, 43007, Spain
5Critical Care Department, Hospital Universitario La Fe, Avda Campanar 21, Valencia, 46009, Spain
6Microbiology Service, Hospital Son Llatzer, Ctra. Manacor, km 4, Palma de Mallorca 07198, Spain
7Intensive Care Unit, Hospital General San Jorge, Avenida Martínez De Velasco 36, Huesca, 22004ý, Spain
8Intensive Care Unit & Internal Medicine Service, Hospital Gregorio Marañón, C/Doctor Esquerdo 46, Madrid, 28007, Spain
9Microbiology Service, Hospital Universitario de Canarias, Carretera Del Rosario 145, Santa Cruz De Tenerifeý, 38009, Spain
10Microbiology Service, Hospital General de La Palma, Buenavista de Arriba, s/n, Breña Alta, 38713, Spain
11Intensive Care Unit, Hospital Universitario de Canarias, Carretera Del Rosario 145, Santa Cruz De Tenerifeý, 38009, Spain
12Intensive Care Unit, Hospital Virgen del Rocío, Avenida Manuel Siurot s/n, Sevilla, 41013, Spain
13Intensive Care Unit Service, Hospital Son Llatzer, Ctra. Manacor, km 4, Palma de Mallorca, 07198, Spain
14Intensive Care Unit Service, Hospital Lozano Blesa, Avenida San Juan Bosco 15, Zaragozaý,50009, Spain
15Microbiology Service, Hospital Universitario Ramón y Cajal & CIBERESP, Carretera Colmenar Viejo KM 9,100, Madrid, 28049, Spain
16Intensive Care Unit, Hospital Universitario Ramón y Cajal, Carretera Colmenar Viejo KM 9,100, Madrid, 28049, Spain
17Infectious Diseases Service, Hospital Clínico Universitario, Avda Ramón y Cajal 3, Valladolid, 47005, Spain
18Preventive Medicine Service, Hospital Universitario Valle Hebron & CIBERESP, Paseo Vall d'Hebron, 119-129, Barcelona, 08035, Spain
19Experimental Theraputics Division, University Health Network, Medical Discovery Tower, 3rd floor Room 913-916,101 Collegue Street, Toronto,
ON M5G 1L7, Canada
20International Institute of Infection and Immunity, Shantou University, 22 Xinling Road, Shantou, Guangdong Province, 515031, PR China
21Intensive Care Unit, Hospital de Villarobredo, Avenida Miguel de Cervantes s/n, Villarrobledo, 02600, Spain
22Department of Immunology, University of Toronto, Medical Discovery Tower, 3rd floor Room 913-916,101 Collegue Street, Toronto, ON M5G 1L7,
Canada
Corresponding author: Jesus F Bermejo-Martin, jfbermejo@saludcastillayleon.es
Received: 4 Nov 2009 Revisions requested: 2 Dec 2009 Revisions received: 3 Dec 2009 Accepted: 11 Dec 2009 Published: 11 Dec 2009
Critical Care 2009, 13:R201 (doi:10.1186/cc8208)
This article is online at: http://ccforum.com/content/13/6/R201
© 2009 Bermejo-Martin et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction Human host immune response following infection
with the new variant of A/H1N1 pandemic influenza virus
(nvH1N1) is poorly understood. We utilize here systemic
cytokine and antibody levels in evaluating differences in early
immune response in both mild and severe patients infected with
nvH1N1.
FGF-b: Human Fibroblast Growth Factor-basic; G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte macrophage colony-stimulating
factor; IFN-α: interferon alpha; IFN-γ: interferon γ; IL-1RA: Interleukin 1 receptor antagonist; IP-10: Interferon-inducible protein-10; MCP-1: monocyte
chemoattractant protein-1; MIP-1α: macrophage inflammatory protein-1α; MIP-1β: macrophage inflammatory protein-1β; nvH1N1; new variant of
H1N1 influenza virus; PDGF-BB: platelet-derived growth factor; TNF-α: tumour necrosis factor α; VEGF: vascular endothelial growth factor.

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Methods We profiled 29 cytokines and chemokines and
evaluated the haemagglutination inhibition activity as
quantitative and qualitative measurements of host immune
responses in serum obtained during the first five days after
symptoms onset, in two cohorts of nvH1N1 infected patients.
Severe patients required hospitalization (n = 20), due to
respiratory insufficiency (10 of them were admitted to the
intensive care unit), while mild patients had exclusively flu-like
symptoms (n = 15). A group of healthy donors was included as
control (n = 15). Differences in levels of mediators between
groups were assessed by using the non parametric U-Mann
Whitney test. Association between variables was determined by
calculating the Spearman correlation coefficient. Viral load was
performed in serum by using real-time PCR targeting the
neuraminidase gene.
Results Increased levels of innate-immunity mediators (IP-10,
MCP-1, MIP-1β), and the absence of anti-nvH1N1 antibodies,
characterized the early response to nvH1N1 infection in both
hospitalized and mild patients. High systemic levels of type-II
interferon (IFN-γ) and also of a group of mediators involved in the
development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper
1 (TNF-α, IL-15, IL-12p70) responses were exclusively found in
hospitalized patients. IL-15, IL-12p70, IL-6 constituted a
hallmark of critical illness in our study. A significant inverse
association was found between IL-6, IL-8 and PaO2 in critical
patients.
Conclusions While infection with the nvH1N1 induces a typical
innate response in both mild and severe patients, severe
disease with respiratory involvement is characterized by early
secretion of Th17 and Th1 cytokines usually associated with cell
mediated immunity but also commonly linked to the
pathogenesis of autoimmune/inflammatory diseases. The exact
role of Th1 and Th17 mediators in the evolution of nvH1N1 mild
and severe disease merits further investigation as to the
detrimental or beneficial role these cytokines play in severe
illness.
Introduction
The emergence of the new pandemic variant of influenza virus
(nvH1N1) has brought renewed attention to the strategies for
prevention, treatment and minimization of the social and
human costs of the influenza disease [1-5]. The great majority
of nvH1N1 infections are mild and self-limiting in nature [6-8].
Nevertheless, a small percentage of the patients require hos-
pitalization and specialized attention in Intensive Care Units
(ICUs) [9-12]. Many severe cases occur in healthy young
adults, an age group rarely seriously affected by seasonal influ-
enza [9-14]. While pregnancy and metabolic conditions
(including obesity and diabetes) have been identified as risk
factors for severe nvH1N1 disease, 40 to 50% of fatal cases
have no documented underlying medical condition [11,12,14].
The new virus causes more severe pathological lesions in the
lungs of infected mice, ferrets and non-human primates than
seasonal human H1N1 virus [15]. The role of host immune
responses in clearance of nvH1N1 or the role, if any, of host
immune responses in contributing to severe respiratory patho-
genesis of nvH1N1 infections is not known at this time. We
have previously identified specific host immune response
chemokine and cytokine signatures in severe and mild SARS
CoV, H5N1 and Respiratory Syncytial Virus infections. In
these studies, early host immune responses are characterized
by the expression of systemic levels of chemokines, such as
CXCL10, indicative of innate anti viral responses [16-19].
Severe and mild SARS and RSV illness could further be
defined by chemokine and cytokine signatures involved in the
development of adaptive immunity. Interestingly, de Jong et al.
have demonstrated that hypercytokinemia of specific chemok-
ines and cytokines is associated with severe and often fatal
cases of human H5N1 infections [20]. To determine if host
immune responses play a potential role in the evolution of mild
or severe nvH1N1 illness we performed an analysis of sys-
temic chemokine and cytokine levels in serum from severe and
mild nvH1N1 patients shortly following the onset of symptoms.
Interestingly, we identified cytokine signatures unique to mild
and severe patients.
Materials and methods
Patients and controls
Both hospitalized and outpatients were recruited during the
first pandemic wave in the months of July and August 2009 in
10 different hospitals within the National Public Health System
of Spain.
Inclusion criteria: Critical patients with respiratory insuffi-
ciency, hospitalized non critical patients with respiratory insuf-
ficiency, and mild outpatients with no respiratory insufficiency
attending to the participant centers with confirmed nvH1N1
infection by molecular diagnostic methods (see below) were
asked to donate a serum sample for the study in the first con-
tact with the participant physicians. Initially we enrolled 35
hospitalized patients and 31 outpatients. To determine sys-
temic levels of chemokines and cytokines in sera from nvH1N1
infected individuals, we analyzed sera from 20 hospitalized, 15
outpatients, and 15 control subjects for levels of 29 different
mediators. The final number of patients used for analysis was
based on exclusion and matching criteria listed in Figure 1.
Exclusion criteria: Patients with signs of bacterial infection
defined by the presence of purulent respiratory secretions,
and/or positive results in respiratory cultures, blood cultures,
and/or positive urinary antigen test to Legionella pneumophila
or Streptococcus pneumoniae were excluded from the analy-
sis (Figure 1). Children under 16 years old and one patient
older than 80 years old were also excluded in order to make
groups comparable by age. Pregnant women were also
excluded to avoid confusion factors during the analysis of the
immune response to the virus, since pregnancy induces phys-
iological changes in the immune system (Figure 1). Informed
consent was obtained directly from each patient or their legal

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representative and also from the healthy controls before enroll-
ment. Approval of the study protocol in both the scientific and
the ethical aspects was obtained from the Scientific Commit-
tee for Clinical Research of the coordinating center (Hospital
Clinico Universitario de Valladolid, Spain).
Samples and laboratory studies
Sample collection and transport
Blood samples were collected by experienced nurses. A sin-
gle serum sample was obtained from each patient or control.
Serum samples were obtained after proper centrifugation and
were sent refrigerated to the National Influenza Center of Val-
ladolid (Spain), where they were stored at -70°C until immune
mediator profiling, haemagglutination inhibition activity (HI)
and viral load evaluation. Nasopharyngeal swabs preserved in
virus transportation medium were sent to the World Health
Organization (WHO) associated National Influenza Centers of
Valladolid, Majadahonda and Barcelona, Spain for viral diag-
nosis purposes.
Viral diagnosis
Viral RNA from nasopharyngeal swabs was obtained by using
automatic extractors (Biomerieux® (Marcy l'Etoile, France),
Roche® (Basel, Switzerland) and viral presence was assessed
by real time PCR based methods using reagents provided free
of charge by the Centers for Disease Control (CDC, Atlanta,
USA) or purchased from Roche® (Basel, Switzerland) (H1N1
detection set) on 96-well plate termocyclers (Roche® LC480
(Basel, Switzerland) and Applied Biosystems® 7500 (Foster
City, CA, USA)
Viral load measurement
Viral load was measured and compared between groups by
real time reverse transcription PCR on RNA extracted from
Figure 1
Flow chart detailing patients' recruitment and sample collectionFlow chart detailing patients' recruitment and sample collection.
Healthy controls (n=15) were recruited between H ealth Care Work ers at the Hospital Clínico
Universitario de Valladolid and their relatives. None of them showed signs of respiratory or other
focality infection or inflammatory conditions at the time of sam ple collection.
Critical patients with
respiratory
insufficiency
admitted to the ICU
with positive PCR for
pandemic H1N1
N=21
Excluded from
analysis:
- Two pregnant
women
- Two children under
16 yrs old
- Seven patients whose
samples were taken
later than 5 days after
hospital admission
plus evidenced
bacterial infection
TOTAL
EXCLUDED = 11
CRITICAL
PATIENTS
Included in the analysis:
Patients with sampling da y in
the first 5 days after hospital
admission and no documented
bacterial infection
Sample collection took place
4.5 [5.0] days after disease
onset
TOTAL INCLUDED = 10
CRITICAL PATIENTS
Non critical patients with
respiratory insufficiency
admitted to the hospital
with positive PCR for
pandemic H1N1
N=14
Excluded from
analysis:
- One 83-yr-old man
- Three patients whose
samples were taken
later than 5 days after
hospital admission
TOTAL
EXCLUDED = 4
PATIENTS
Included in the analysis:
Patients with sampling da y in
the first 5 days after hospital
admission and no documented
bacterial infection.
Sample collection took place
2.0 [1.2] days after disease
onset
TOTAL INCLUDED = 10
PATIENTS
Outpatients with no
respiratory insufficiency
with positive PCR for
pandemic H1N1
N= 31
Excluded from
analysis:
- Sixteen children
under 16 yrs old
TOTAL
EXCLUDED = 16
PATIENTS
Included in the analysis:
Patients with sampling da y in
the first contact with health
services and no documented
bacterial infection.
Sample collection took place
3.0 [2,7] days after disease
onset
TOTAL INCLUDED = 15
PATIENTS

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serum. Briefly, an external curve was obtained by using a serial
dilution of human RNA extracted from cultured monocytic
leukemia (THP-1) cells, and human gene GAPDH was
employed as reporter gene. nvH1N1 neuraminidase gene was
amplified by QRT-PCR in each serum sample, and crossing
points were extrapolated to the external curve. Analysis of
samples and standard curve was conducted by using the
7500 fast v2.0.3 software (Applied Biosystem™). Results
were given as relative comparisons in (pg RNA/μl). 5'-3'
sequences of primer pairs: GAPDH 5'-ACCCAGAAGACT-
GTGGATGG-3' (forward); 5'-TTCTAGACGGCAGGT-
CAGGT-3' (reverse); nvH1N1 neuraminidase: 5'-
TCAGTCGAAATGAATGCCCTAA-3' (forward) and N1R 5'-
CACGGTCGATTCGAGCCATG-3'(reverse).
Cytokines and chemokines quantification
Serum chemokine and cytokine levels were evaluated using
the multiplex Biorad© 27 plex assay (Hercules, CA, USA). This
system allows for quantitative measurement of 27 different
chemokines, cytokines, growth-factors and immune mediators
while consuming a small amount of biological material. Further-
more, this system has good representation of analytes for
inflammatory cytokines, anti-inflammatory cytokines, Th1
cytokines, Th2 cytokines, Th17 cytokines and chemokines,
allowing for the testing of differential levels of regulatory
cytokines in the serum of severe and mild patients. Addition-
ally, interferon α, adiponectin and leptin were measured by
using an enzyme-linked inmuno adsorbant assay (ELISA) from
R&D© Systems (Minneapolis, MN, USA).
Haemagglutination inhibition assay (HI)
HI assays were performed on a 100 μl aliquot of the samples
at University Health Network (UHN), Toronto, Ontario, Can-
ada. The sera was treated with Receptor-Destroying Enzyme
(RDE) of V. cholerae by diluting one part serum with three
parts enzyme and were incubated overnight in a 37°C water
bath. The enzyme was inactivated by a 30-minute incubation
at 56°C followed by the addition of six parts 0.85% physiolog-
ical saline for a final dilution of 1/10. HI assays were performed
in V-bottom 96-well microtiter plates (Corning Costar Co.,
Cambridge, MA, USA) with 0.5% turkey erythrocytes, as pre-
viously described [21], using inactivated pandemic influenza
A/California/07/2009 (nvH1N1) antigens.
Statistical analysis
Data analysis was performed using SPSS 15.0. Comparisons
between groups were performed using the non parametric U-
Mann Whitney test. Data are displayed as (mean, standard
deviation) for clinical and laboratory parameters and as
(median, interquartile rank) for data on sample collection tim-
ing and the immune mediators levels. Association between
variables was determined by calculating the Spearman corre-
lation coefficient (r) and data shown as (r, P value). Signifi-
cance was fixed at P value < 0.05
Results
Patient's characteristics
All the patients showed symptoms of acute respiratory viral
infection at disease onset. The most frequent initial symptoms
were (% of patients in each group: critical, hospitalized non
critical, outpatients): fever (100, 100, 80), cough (100, 90,
80), headache (90, 80, 40), tiredness (100, 80, 66) and myal-
gia (50, 80, 46). Hospitalized patients showed dyspnoea as
the initial symptom in 90% of the cases and 100% developed
respiratory insufficiency at the time of hospital admission (dys-
pnoea and/or hypoxemia defined as O2 saturation < 95%
breathing at least two liters of oxygen). Ten patients required
admission to an intensive care unit (ICU) due to their respira-
tory situation. The remaining 10 were admitted to other differ-
ent specialized hospital services. Outpatients had no
difficulties with respiratory function, showing respiratory rates
under 25×'. Sex composition was the same for both critical
and non critical hospitalized patients: 60% of the patients
were male (n = 12) and 40% female (n = 8). Fifty-three per-
cent of the outpatients were male and 47% female (n = 8 and
7 respectively) (Table 1). Average age was as follows: hospi-
talized patients (36.6; 11.5), outpatients (29.7; 8.0) and
healthy controls, (29.5; 13.2). Critical patients were slightly
older than the other hospitalized patients (Table 1). Seven
patients with critical illness and four severe patients with non
critical illness showed previous pathologies (Table 1). Ten out
of 10 of the critical patients, and 6/10 of the severe non critical
patients showed a pathological chest x-ray within 24 hours of
onset of the symptoms (Table 1). Outpatients had received
just antipyretics (paracetamol) before sample collection (none
of them had received oseltamivir). One hundred percent of the
hospitalized patients (critical and non critical), had received
oseltamivir at the time of sample collection (Table 1). Lympho-
penia was a common finding in the critical patients (mean; SD)
(358.5; 267.1). LDH levels were increased over normal levels
in hospitalized patients, mostly in those critically ill (Table 1).
Furthermore, critical patients also showed high levels of CPK,
GOT, GPT and glucose in venous blood (Table 1). Critical
patients stayed longer at the hospital than the other hospital-
ized patients (Table 1). Three critical patients ultimately died
(five days after onset due to hypoxemia and septic shock; 69
days after onset by refractory hypoxemia complicated by sys-
temic candidiasis; and the third after 75 days of supportive
therapy by multiorganic failure).
HI activity
HI activity (A/California/07/2009) was present in serum from
only two critically ill patients of 50 and 51 years old (titres 1/
1280 and 1/160 respectively) and in one 25-year-old outpa-
tient (titre 1/160). Serum from those three patients showing HI
showed also the ability to block viral replication, as assessed
by microneutralization assay against A/California/07/2009
(data not shown). This data supports the notion that at the time
of sampling the vast majority of the patients had yet to produce

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Table 1
Clinical and laboratory characteristics of the patients
Hospitalized, critical illness(n = 10) Hospitalized, non critical illness (n = 10)
Pathological antecedents
Esquizophrenia 1/10 -
COPD 1/10 -
Diabetes 2/10 -
Asthma - 2/10
COPD+HIV - 1/10
Chronic disease conective tissue - 1/10
Dyslipemia 1/10 -
Cardiopathy 1/10 -
Hypertension 1/10 -
Obesity (BMI>30) 5/10 3/10
Descriptives
Age (yrs) 41.8 (9.9) 31.3 (10.9)
Sex (M/F) 6/4 6/4
Days at hospital 29.5 (29.2) 6.5 (2.8)
Days at ICU 26.6 (30.8) 0 (0)
Severity scores
SOFA score 5.6 (2.9) -
APACHEII score 12.8 (4.2) -
Respiratory condition
Mechanical Ventilation 9/10 0/10
O2 saturation (%) 83.3 (7.3) 93.0 (5.1)
PaO2 (mmHg) 54.1 (11.6) 76.5 (24.3)
PaO2:FiO2 94.0 (89.9) 252.5 (20.6)
Opacity in initial chest X-Ray
0/4 quadrants 0/10 4/10
1/4 quadrants 2/10 3/10
2/4 quadrants 3/10 3/10
3/4 quadrants 0/10 0/10
4/4 quadrants 5/10 0/10
Biochemistry
LDH (IU/liter) 1634 (1226.0) 475.1 (356.7)
CPK (IU/liter) 588.5 (606.1) 108.5 (132.1)

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antibodies against nvH1N1 and was in the early stages of dis-
ease.
Immune mediators profiling
The virus induced in both mild and severe patients a systemic
elevation of three chemokines that have been shown to be
expressed early during viral infections, CXCL-10 (IP-10),
CCL-2 (MCP-1) and CCL-4 (MIP-1β), with no differences in
the levels of these mediators between them (data on immune
mediators profiling are shown in Figure 2 and Additional file 1).
IL-8, IFN-γ, IL-13, IL-10 levels were higher in the hospitalized
patients than in outpatients and controls (P < 0.05). IL-9
behaved in a similar way. While both critical and non-critical
hospitalized patients showed higher levels of IL-17 and TNF-α
than controls, only severe non critical patients showed signifi-
cant higher levels of IL-17 and TNF-α than mild. On the other
hand, IL-15 and IL-12p70 increased exclusively in critical
patients, who in addition showed the highest levels of IL-6 of
the compared groups.
To determine if systemic viral load plays a role in chemokine or
cytokine expression levels we evaluated serum for nvH1N1
levels. Fifty-seven percent of critical patients, 50% of hospital-
ized non critical patients, and 93% of mild patients showed
positive virus in serum. For those with positive virus in serum,
we found no differences in viral load between critical patients,
hospitalized non critically ill, and mild outpatients (Figure 3).
We found significantly higher levels of IL-13 and IL-17 in those
hospitalized patients with negative virus in serum compared to
those with virus in serum (data not shown). Similarly, inverse
correlations were found between viral load and IL-13, IL-17 in
patients requiring hospital admission (Figure 4). When media-
tor levels were correlated with the clinical parameters, a signif-
icant inverse association was found between IL-6 and PaO2
in hospitalized patients (Figure 4). Exclusively in the critical
patients group, IL-8 inversely correlated with PaO2 [-0.7;
0.028]. In the non critically ill hospitalized patients group, a
negative association was observed between IL-15 and PaO2
[-0.7; 0.039].
Discussion
In a first attempt to understand the role host immune
responses play in the evolution of severe and mild nvH1N1
disease, we assessed systemic levels of chemokines and
cytokines in the sera from hospitalized and outpatients. Con-
sistent with our previous studies on early elevated expression
of CXCL10, CCL2 and CCL4 in SARS CoV and RSV infected
patients [16-19], we found in the present study elevated
expression of these chemokines in severe patients (critical and
non critical) and mild patients. The early expression of these
chemokines in all patients likely is indicative of innate antiviral
host responses.
One of the most intriguing observations in our present study is
the dramatic increase of mediators which stimulate Th-1
responses (IFN-γ, TNF-α, IL-15, IL-12p70) and Th-17 ones (IL-
8, IL-9, IL-17, IL-6) in the severe patients (Figure 5). Th-1 adap-
tive immunity is an important response against intracellular
microbes such as viruses [22]. Th-17 immunity participates in
clearing pathogens during host defense reactions but is
involved also in tissue inflammation in several autoimmune dis-
eases, allergic diseases, and asthma [23-27].
GOT (U/liter) 126.9 (73.5) 35.6 (14.5)
GPT (U/liter) 130.7 (97.6) 35.7 (18.9)
Glucose (mg/dl) 202.7 (97.1) 113.1 (29.1)
CRP (mg/l) 85.4 (76.3) 61.1 (105.1)
Treatment received at the time of sample collection
Oseltamivir 10/10 (75-150 mg/12 hs) 10/10 (75 mg/12 hs)
Cephalosporines 6/10 2/10
Macrolides 3/10 1/10
Quinolones 5/10 7/10
Steroids 4/10 (parenteral) 2/10 (inhaled)
Noradrenaline 5/10 0/10
Renal replacement therapy 2/10 0/10
COPD = Chronic Obstructive Pulmonary Disease; HIV = Human Immunodeficiency Virus; BMI = Body Mass Index; ICU = Intensive Care Unit;
SOFA = Sepsis-related Organ Failure Assessment score; APACHE II = Acute Physiology and Chronic Health Evaluation II; PaO2 = pressure of
oxygen in arterial blood; FiO2 = fraction of inspired oxygen; LDH = Lactate dehydrogenase; CPK = creatine phosphokinase; GOT = Glutamyl
oxaloacetic transaminase; GPT = Glutamyl pyruvic transaminase; CRP = C Reactive Protein; IU = International Units; U = Units.
Table 1 (Continued)
Clinical and laboratory characteristics of the patients

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Increase in IFN-γ IL-8, IL-9, IL-13 and IL-10 in both critical and
non critical hospitalized patients compared to mild ones indi-
cates that they constitute hallmarks of severe disease. IFN-γ
and IL-8 promote antiviral immunity but also respiratory tract
inflammation by recruiting neutrophils and mononuclear cells
to the site of the infection [28-30]. IL-9 is a Th2 cytokine that
induces differentiation of Th-17 cells [26]. IL-10 and IL-13
show immunomodulatory properties. IL-13 attenuates Th-17
cytokine production [31]. IL-10 is known to be an anti-inflam-
matory cytokine. In a murine model, McKinstry et al.revealed
that IL-10 inhibits development of Th-17 responses during
influenza infection, correlating with compromised protection
[32]. Increase of IL-17 and TNF-α in hospitalized patients over
control indicated that they also parallel severe disease, but the
significantly higher levels of IL-17 and TNF-α in severe non
critical patients compared to mild (difference not found for crit-
ical ones), could reflect a beneficial role of these cytokines in
this particular subset of patients. The patient who died five
days after disease onset showed high viral load and undetec-
table IL-17 levels in serum. This could reflect a protective role
of IL-17 in severe patients. IL-15, IL-12p70, IL-6 constituted a
hallmark of critical illness in our study. These three cytokines
also mediate both antiviral and pro-inflammatory responses. IL-
6 is a potent regulator switching immune responses from the
induction of Foxp3+ regulatory T cells to pathogenic Th17
cells in vivo [33]. IL-15 promotes CD8 T cells homeostatic
proliferation [34] in response to infection. IL-12 plays a key
role in the switch from innate to adaptive immunity [17].
Figure 2
Levels of immune mediators in the four groupsLevels of immune mediators in the four groups. *Significant differences with control at the level P < 0.05.
*
* *
*
*
*
*
*
*
*
**
*
*
*
*
*
*
*

Critical Care Vol 13 No 6 Bermejo-Martin et al.
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High levels of Th-1 and Th-17 related mediators could support
the hypothesis of a Th-1+Th-17 inflammatory response in the
origin of the severe respiratory disease caused by nvH1N1
infection. Alternatively, an increase in Th-1 and Th-17
cytokines may reflect a vigorous antiviral host response neces-
sary for clearance of virus during severe lower respiratory
infections. While the ability of influenza A virus to induce the
production of chemotactic (RANTES, MIP-1α, MCP-1, MCP-
3, and IP-10) and pro-inflammatory (IL-1β, IL-6, IL-18, and
TNF-α) Th1 related mediators is well know from previous
reports on seasonal influenza [29,35], this is the first report
evidencing Th17 response as a signature of severe influenza
disease in humans [36,37]. Since there are immunomodula-
tory drugs which have shown to down-modulate the activity of
both Th1 and Th17 [38], the results obtained here supports
the development of further studies on animal models aimed to
clarify the role of these mediators in the pathogenesis of the
acute respiratory disease showed by severe nvH1N1 infected
patients.
Conclusions
Analysis of the immune mediators involved in host responses
to the virus in mild and severe cases revealed Th1 and Th17
cytokine responses as early distinctive hallmarks of severe res-
piratory compromise following infection with nvH1N1. The
exact role of Th1 and Th17 mediators in the evolution of
nvH1N1 mild and severe disease merits further investigation
as to the detrimental or beneficial role these cytokines play in
severe illness. The influence of Th17-dominant conditions
(autoimmune diseases) or Th1 deficient ones (HIV infection)
on disease outcome should also be explored. Furthermore, the
impact of other regulatory cytokines elevated in severe dis-
ease (IL-10, IL-13) on the evolution of host immune responses
to nvH1N1 infections may represent alternative therapeutics
for controlling severe illness.
Figure 3
Viral load in serumViral load in serum. (From left to right: 0: critical patients; 1: hospitalized
(non critical) patients; 2: mild outpatients). Results are expressed as
(pg RNA/μl).
Figure 4
Correlation studiesCorrelation studies. From left to right: correlation between IL-13 level and viral load in serum; correlation between IL-17 level and viral load in serum;
correlation between IL-6 serum levels and PaO2.
Ͳ Ͳ Ͳ 

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Page 9 of 11
(page number not for citation purposes)
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TP, JR and IML assisted in the design of the study, coordi-
nated patient recruitment, analysed and interpreted the data,
and assisted in writing the paper. PR, MCG, CS, DM, JMG,
SH, ES, MG, AC, BV, CJL, JAD, CH, IG and PC supervised
clinical aspects, participated in patient recruitment, assisted in
the analysis, interpretation of data, and writing the report. AT,
MJR, MLM, VF, MAM, MRD, JCG, RC, SR and JME performed
viral diagnosis, assisted in the analysis, interpretation of data,
and writing the report. RA performed cytokine profiling, and
assisted in supervision of laboratory work and writing the
report. NT collected clinical data, and assisted in writing the
report. TR, DB performed the HAI assays and assisted in writ-
ing the report. DV and AL designed and performed the quan-
titative PCR method for viral load measurement. JFBM, DJK
and ROL were the primary investigators, designed the study,
coordinated patient recruitment, supervised laboratory works,
and wrote the article.
Additional files
Key messages
• The great majority of infections caused by the new influ-
enza pandemic virus are mild and self-limiting in nature.
Nevertheless, a small percentage of the patients
develop severe respiratory disease. Analysis of the
immune mediators involved in host responses to the
virus along with the evaluation of the humoral responses
in mild and severe cases may help understand the path-
ogenic events leading to poor outcomes.
• Early response to the virus in both hospitalized and out-
patients was characterized by expression of chemok-
ines (CXCL10, CCL2 and CCL4), also observed in the
response to SARS CoV, H5N1 and RSV, which previ-
ous literature describes to correspond to innate antiviral
responses.
• Patients who develop respiratory compromise in the
first days following infection with nvH1N typically
showed Th1 and Th17 hyper-cytokinemia, compared to
mild patients and healthy controls. These cytokine pro-
files have been previously reported to participate in both
antiviral and pro-inflammatory responses.
• Increased systemic levels of IL-15, IL-12p70, IL-6 con-
stituted a hallmark of critical illness. These mediators
are known to promote the development of adaptive
responses and also pro-inflammatory ones in other viral
infections.
• Our findings constitute a major avenue to guide the
design of further works studying the beneficial or detri-
mental role of Th1 and Th17 responses in this disease.
The following Additional files are available online:
Additional file 1
Table listing the immune mediators' profiles in serum
during the early response against the nvH1N1 virus.
See http://www.biomedcentral.com/content/
supplementary/cc8208-S1.doc
Figure 5
Predominant cytokine profiles paralleling early nvH1N1 disease by clinical severityPredominant cytokine profiles paralleling early nvH1N1 disease by clinical severity.
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Critical Care Vol 13 No 6 Bermejo-Martin et al.
Page 10 of 11
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Acknowledgements
This work has been made by an international team pertaining to the
Spanish-Canadian Consortium for the Study of Influenza Immuno-
pathogenesis. The authors would like to thank Lucia Rico and Verónica
Iglesias for their assistance in the technical development of the multiplex
cytokine assays, to Begoña Nogueira for her technical support, and to
Nikki Kelvin for language revision of this article. This work was possible
thanks to the financial support obtained from the Ministry of Science of
Spain and Consejería de Sanidad Junta de Castilla y León, Programa
de investigación comisionada en gripe, GR09/0021, Programa para
favorecer la incorporación de grupos de investigación en las Instituci-
ones del Sistema Nacional de Salud, EMER07/050, and Proyectos en
Investigación Sanitaria, PI081236. CIHR, NIH and LKSF-Canada sup-
port DJK. This sponsorship made possible reagent acquisition and sam-
ple transportation between participant groups.
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