heat stress can negatively affect an animal 's growth performance and the immune competence to some bacterial or viral infection     [ 16 ] . it has been reported that heat stress results in decease of weights of both primary and secondary lymphoid organs , profiles of circulating leukocytes , t cell in the blood , and antibody response to sheep red blood cells ( srbcs ) or against newcastle disease     [ 710 ] . our previous studies have demonstrated that chronic heat stress condition negatively affects both humoral and cellular responses against foot and mouth disease virus ( fmdv ) in mice . however , there have been few detailed studies addressing the effects of chs on the innate immune response as the most immediate defense against viral infection . the h5n1 subtype of highly pathogenic avian influenza viruses ( hpaivs ) causes infections in domestic poultry and humans . it is epidemiologically characterized by wide dissemination and rapid prevalence and is a threat to public health . so far human cases of h5n1 infection in worldwide have increased to 522 , including 309 deaths ( http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_02_25/en/index.html ) . the data from who demonstrated that most human cases of h5n1 infection occurred in the tropical and subtropical countries , such as egypt , vietnam , and indonesia , instead of the high latitude countries . ( http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_02_25/en/index.html ) . in mainland china , the outbreak of h5n1 virus in poultry and human cases mainly occurred in the south of the yangtze associated with high humidity and temperature , including guangdong , guangxi and , fujian province . it is speculative that high temperature may be associated with human or poultry higher susceptibility to h5n1 virus . it was also reported that the vast temperature changes frequently occurred one week before the avian influenza outbreak in china . even in winter , heat stress often occurred under conditions of crowding , heating , and without ventilation in poultry . when those flocks are exposed to h5n1 virus , the outbreaks of avian influenza may occur . in this study , we measured the effects of chs on innate host immunity by detecting local immunity in the respiratory tract , maturation or activation of dcs , and cytokine levels in spleens of the mice under chs or tn conditions . then we examined mortality rate , histopathology , and viral loads in lung of chs mice challenged with h5n1 virus . we demonstrated that heat stress could increase the susceptibility of animals to the highly pathogenic avian influenza virus ( hpaiv ) h5n1 . the h5n1 influenza virus ( a / chicken / henan/1/04 ) used in this study was isolated from infected chicken flocks . this isolate was highly pathogenic on poultry , mouse , and madin - darby canine kidney ( mdck ) cells . the virus has been adapted in mdck cells for convenience and propagated in cell culture at 37c for 48 hours . female balb / c mice that were 810-weeks - old were obtained from vital river laboratories ( beijing , china ) , while the original breeding pairs were purchased from charles river ( beijing , china ) . mice were raised in independent ventilated cages and received pathogen - free food and water . experimentation with animals was governed by the regulations of experimental animals of beijing authority and approved by the animal ethics committee of the china agriculture university . mice in the chs group were placed in a biological oxygen demand ( bod ) incubator for 21 days and subjected to chronic heat exposure for 4  h at a temperature of 38  1c , simulating high summer temperatures . mice in the tn and control groups were kept in an incubator at 24  1c to simulate room temperature . mice were sacrificed at various time - points and the lung and spleen from each mouse were collected . mice were anesthetized with zotile ( virbac , france ) intramuscular at a dose of 15  mg / kg ( body weight ) and were then infected intranasally with 50 plaque forming units ( pfus ) of h5n1 virus . mice were sacrificed at various time - points and the lung and spleen from each mouse were collected and stored in liquid nitrogen until required . mdck cells were cultured in dulbecco 's modified eagle 's medium ( dmem ; hyclone laboratories ) containing 10% fbs ( hyclone laboratories ) , 100  u / ml penicillin , and 100  g / ml streptomycin . the supernatants of mouse lung tissues were diluted 10-fold and added to a monolayer of mdck cells in semisolid agar that contained 0.5  g / ml of trypsin tpck ( sigma - aldrich , st louis , mo , usa ) . cultures were incubated at 37c , 5% co2 for 6072 hours , fixed , and stained with 1% crystal violet . tissues were removed and fixed with 4% neutral formalin at room temperature for 48  h. the serial tissue sections were cut to 5  m thickness after embedding in paraffin . each slide was stained with hematoxylin and eosin ( h&e ) and then examined by light microscopy ( olympus cx31 ) . sections were incubated in 10% normal goat serum in pbs for 30 minutes to block non - specific binding sites before being reacted with the anti - influenza nucleoprotein mab ( aa5h , abcam ) at 1  :  1000 dilutions in pbs for 2  h. the slides were further incubated with goat anti - mouse igg conjugated with avidin ( chemicon , usa ) for 1  h and followed by an incubation of the biotinlated peroxidase ( victoria , bc , canada ) for additional 1  h. staining was visualized by the addition of 3,3-diaminobenzidin ( dab , sigma , st . , usa ) for 15  min and counterstained with haematoxylin mounted with neutral balsam . total rna was prepared from 10  mg lung tissue homogenized in trizol ( invitrogen , carlsbad , ca , usa ) according to the manufacturer 's instructions . the dnasei - treated rna ( 0.2  g ) quantification of expression of the hemagglutinin ( ha ) gene of h5n1 influenza virus was conducted using a power sybr green pcr master mix kit ( abi ) . the following primers were used in the qpcr : forward primer , 5-cgc agt att cag aag aag caa gac-3 ; and reverse primer , 5-tcc ata agg ata gac cag cta cca-3. the reaction was run on an abi 7500 thermal cycler with an initial denaturation step at 95c for 10 minutes , followed by 40 cycles of 95c for 15 seconds , 56c for 30 seconds , and 72c for 40 seconds . the copy number of the ha gene was calculated using an ha - containing plasmid of known concentration as a standard . quantification by qpcr was performed for five other genes : -actin ( primers , 5-gag acc ttc aac acc ccg c-3 and 5-atg tca cgc acg att tcc c-3 ) , il-6 ( 5-agc cag agt cct tca-3 and 5-tct tgg tcc tta gcc-3 ) , il-10 ( 5-ggt tgc caa gcc tta tcg ga-3 and 5-acc tgc tcc act gcc ttg ct-3 ) , ifn- ( 5-tcc agc tcc aag aaa gga cg-3 and 5-gca tct tct ccg tca tct cc-3 ) , and hsp70 ( 5-tga gca gcc cat cct tag tg-3 and 5-ata ggc atc cgt ccc ttt gt-3 ) . reactions were carried out on an abi 7500 with initial denaturation at 95c for 10  min , then 40 cycles of denaturation at 95c for 15  s , annealing at 50c for 30  s , and extension at 72c for 40  s using a power sybr green pcr master mix kit . gene expression was normalized to that of the tn group using the 2 method with -actin as an internal standard . at 21 days after exposure to 38 1c or 24  1c , the mice were euthanized with a lethal dose of pentobarbital . the bal collected from one mouse was pooled centrifuged , and cells were resuspended in 100  ml saline containing 0.1% bsa . two hundred cells were examined per slide for the pulmonary alveolar macrophage ( pam ) count . fluorophore - conjugated rat antimouse monoclonal antibodies including anti - mhc - ii - pe , anti - cd40-pe , anti - cd80-pe , anti - cd86-pe , anti - cd11c - fitc , and isotype controls were purchased from bdpharmingen ( san diego , ca , usa ) . three mice from each group were sacrificed after exposure to 38  1c or 24  1c for 21 days , and single cell suspensions from the spleens at a concentration of 2  10  cells/200  l were blocked with 2  l fc monoclonal antibody ( 0.5  g / ml ) for 30  min at 4c . after rinsing once with pbs , cells were stained with isotype controls , or double stained with anti - cd11c - fitc and anti - cd40-pe , or anti - cd11c - fitc and anti - cd80-pe , or anti - cd11c- fitc and anti - cd86-pe , or anti - cd11c - fitc and anti - mhc - ii - pe . the fluorescent intensities were measured with a facscalibur and analyzed with cell questpro software ( bd biosciences , usa ) . statistical analysis was performed using one - way anovas with spss 12.0 ( spss taiwan corp . , to determine the effect of chs on local immunity in the respiratory tract of mice , the histopathological changes in the nasal mucosa , trachea , and lungs of the mice in each group after exposure to 38  1c or 24  1c for 21 days were examined ( figure 1(a ) ) . the nasal mucosa lesions of mice in the chs group exhibited sebaceous gland hyperplasia ( figure 1(a)(b ) ) . the tracheal lesions in the chs mice were characterized by a reduction in tracheal epithelial cells and many erythrocytes had infiltrated the tube cavity of the trachea ( figure 1(a)(d ) ) . changes in lung pathology included a reduction and necrosis of mucous epithelium cells in the bronchioles and alveolar expansion ( figure 1(a)(f ) ) . in comparison , the nasal mucosa , trachea , and lungs from the tn group exhibited no apparent histological changes ( figures 1(a)(a ) , ( c ) , and ( e ) ) . in a previous study , heat stress was characterized by the production of hsps , especially hsp70 . to determine the mrna expression of hsp70 , we used qpcr analysis . hsp70 expression in the lungs of mice was induced at one day after exposure to 38  1c , peaking at day 5 and then returned to normal levels at day 21 ( figure 1(b ) ) . the bal from the mice following exposure to 38  1c or 24  1c for 21 days was collected for pam counting . the number of pams in the chs group were significantly lower than those in the tn group ( figure 1(c ) ) . these results suggest that chs can reduce local immunity in the respiratory tract of mice and also inhibit expression of hsp70 . the maturation or activation of dcs is very important for innate immunity and subsequent adaptive immunity . the expression of costimulatory markers cd40 , cd86 , and cd80 at the dc surface correlated with their capacity to induce or suppress immune responses and the mhc - ii expression is important in the initiation of immune response     . dc maturation of mice exposed to 38  1c or 24  1c for 21 days was determined by facs ( figures 2(a ) and 2(b ) ) . the mhc - ii , cd40 , cd80 and cd86 expression levels in the chs group were lower than these of the tn group , especially cd86 and cd40 ( p < .05 ) . previous studies have shown that il-6 , il-10 , and ifn- played critical roles in the innate immune responses     [ 1820 ] . to determine the effect of chs on the innate immunity associated with cytokine expression in mice , the mrna levels of il-6 , il-10 , and ifn- in the spleen after exposure to 38  1c or 24 the il-6 and ifn- mrna levels of the chs group were downregulated significantly compared to the tn group , while il-10 was significantly upregulated ( p < .05 ) ( figure 2(c ) ) . the mrna level of tnf- was also measured , but there was no significant difference between two groups ( data not shown ) . these data suggest that chs could adversely affect the maturation of dcs and innate immunity in mice .   to determine whether chs could impact upon hapiv h5n1 infection , the mice exposed to 38  1c or 24  1c for 21 days were challenged with 50 pfu of h5n1 virus . the lung tissues of five mice per group were collected at day 3 postinfection for real - time pcr , plaque assay , and histopathogic analysis . results showed that the mice in the chs group had a higher mortality rate ( 67% ) than that in tn group ( 33% ) ( figure 3(a ) ) , but not statistically significant by log rank analysis possibly due to the small sample size . to examine the viral load in the lung of the mice infected with the virus , qpcr and plaque assay were performed . pulmonary viral load in the mice of the chs group was significantly higher than that of tn group at day 3 postinfection ( figures 3(b ) and 3(c ) ) . the h5n1 viral antigen ( np ) was detected extensively in the deciduous alveolar cells and mucosal epithelium cells in the lung ( figure 3(e ) ) . compared with tn group , more h5n1 viral antigen positive cells could be found in the lung of mice after chs treatment . and the results were consistent with the ones of qpcr and plaque assay . the multiple functions of inflammation induced by h5n1 infection potentially affect virus clearance from the host . to evaluate the effect of chs on inflammation in infected mice , expression of cytokines il-6 , ifn- , and il-10 in the lung was determined by qpcr at day 3 postinfection . the mrna levels of il-6 and ifn- in the mice of the chs group were significantly lower than those in the tn group ( figure 3(d ) ) . the soluble cytokine levels in serum of il-6 and ifn- were also measured by elisa , and the results were in similar trend to the mrna levels ( data not shown ) . the results did not show significant differences between two groups ( figure 3(d ) ) . these data indicated that chs could moderately increase the mortality rate and viral load in the lungs of h5n1-infected mice by decreasing il-6 and ifn- expression . to determine the degree of lung lesion in the infected mice , histopathological changes in the lungs of mice in each group at day 3 postinfection were examined ( figure 4 ) . the lungs of the chs group mice showed severe alveolitis , peribronchiolitis , and bronchopneumonia , that was characterized by interstitial edema and inflammatory cellular infiltration around small blood vessels , reduction and necrosis of the mucous epithelium of bronchioles , thickening of alveolar walls , and alveolar lumens flooded with edema fluid mixed with exfoliated alveolar epithelial cells , erythrocytes , and inflammatory cells ( figure 4(c ) ) . the trachea of mice in the chs group exhibited lesions , characterized by exfoliated trachea mucous epithelium cells and a number of erythrocytes and inflammatory cells infiltrated in the tube cavity of the trachea ( figure 4(d ) ) . in mice of the tn group , the lung and trachea lesions were dramatically milder than those observed in the chs group . the lung lesions in the tn group mice showed peribronchiolitis with interstitial edema and inflammatory cell infiltration around small blood vessels , along with edema and reduction of mucous epithelial cells of the bronchioles ( figure 4(a ) ) . the trachea lesions of mice in the tn group mainly demonstrated erythrocyte infiltration ( figure 4(b ) ) . the results suggested that exposure to 38  1c for 21 days aggravated lung and trachea lesions in h5n1-infected mice . in a previous study , we reported that chs could retard the adaptive immune response by suppressing both humoral and cellular responses     . in this study we have demonstrated that chs can significantly suppress host innate immunity in mice and moderately increase the mortality of h5n1-infected mice . chs retarded systemic and local innate immune responses significantly , including the number of pams , dc maturation , mrna levels of il-6 , ifn- , and hsp70 , and caused respiratory system lesions . when mice were infected with h5n1 virus after chs conditioning , the mortality rate and viral load in the lungs increased . lung histopathology results of infected mice further confirmed that chs could moderately increase virulence in h5n1-infected mice . the function and immunity of the respiratory system was closely related to mucosal architecture , pams , and other local immune cells or proteins . in the present study , we showed that chs could reduce local immunity in the respiratory tract of mice by destroying the structural integrity of nasal mucosa , trachea , and lungs and reducing the number of pams . the results suggested that chs could retard the local immunity in respiratory system in mice and rendered them to be susceptible to secondary infection of bacteria or viruses . the hsps , in particular hsp27 , hsp32 , hsp60 , and hsp70 , have an important cytoprotective role during lung inflammation and injury , as they can activate the innate immune system , linking innate and adaptive immune responses     [ 21 , 22 ] . previous studies also indicated that hsps played an immunoregulatory role in chronic inflammation     [ 23 , 24 ] . the results of the expression of hsp70 under chronic heat stress in this study showed that the hsp70 level increased on day 1 , peaked on day 5 , decreased from day 10 , and appeared to be normal level on day 21 after exposure to 38  1c , which suggested that the lack of hsp70 may be associated with the suppression of innate immunity by chs . the dcs are the most potent antigen presenting cells ( apcs ) , and activation of dcs is a crucial and early event of immune regulation     . the expression of costimulatory molecules ( cd40 , cd86 , and cd80 ) at the dc surface correlated with their capacity to induce or suppress immune responses . the mhc - ii molecules expressed on the surface of apcs are important in the initiation of an immune response     . in this study , we observed lower levels of expression of mhc - ii , cd40 , cd80 , and cd86 in chs mice compared to the tn mice , which indicated that chs weakened antigen processing not only in apcs but also in the co - stimulatory pathway , which in turn leads to lower immune responses . il-6 and ifn- play critical roles in the development and maintenance of innate immune responses     [ 18 , 19 , 26 ] . il-10 has been considered to be a suppressive cytokine known to inhibit th1 cytokine expression     [ 20 , 27 , 28 ] . we have shown that chs downregulates il-6 and ifn- expression and upregulates the levels of il-10 . the h5n1 influenza virus can cause severe respiratory symptoms in humans with clinical manifestations that include fever , diarrhea , viral pneumonia , encephalitis , and acute respiratory distress syndrome . high levels of proinflammatory cytokines , including tnf- , il-6 , and cc chemokine ligand 2 ( ccl2 ) , have been detected in human cells and mice infected with highly pathogenic h5n1 influenza virus     [ 29 , 30 ] . generally , a virus - induced cytokine storm has been widely hypothesized to be the main cause of pathology and death during h5n1 infection . there are numerous reports regarding the use of therapeutic agents to control inflammatory responses in influenza infection ; however , it is not clear as to whether they are effective in controlling virus replication . without antiviral agents , knockout mice deficient in tnf- , tnf- receptor 1 , tnf- receptor 2 , il-6 , chemokine ( c - c motif ) ligand 2 , mip-1 , and il-1r or steroid - treated wild - type mice did not have a survival advantage over wild - type mice following influenza virus challenge     [ 32 , 33 ] . on the other hand , type i interferon response that contributes to control of h5n1 virus replication has been demonstrated     [ 34 , 35 ] . the multiple functions of inflammation and cytokines are essential for virus clearance from the host . the antiviral immune response induced by h5n1 virus infection was complicated , involving multiple classes of pattern recognition receptors ( prrs ) , and different signaling pathways . although the inflammation is widely suggested to be the main cause of death from h5n1 infection , the results from the knockout mice deficient in single cytokine were insufficient to clarify the complex process . in this study this may be one of the underlying causes of increased death rates by h5n1 virus after chs treatment . in summary , the data presented here demonstrate that chs treatment significantly suppressed innate host immunity in mice and increased mortality following h5n1 infection . this study is the first to show that chs affects systemic and local innate immune responses as well as survival of h5n1-infected in mice . our results suggest that after a long period of suffering elevated temperatures , the balance in the immune system is destroyed and h5n1 virulence is increased .
chronic heat stress ( chs ) can negatively affect immune response in animals . in this study we assessed the effects of chs on host innate immunity and avian influenza virus h5n1 infection in mice . 
 mice were divided into two groups : chs and thermally neutral ( tn ) . 
 the chs treatment group exhibited reduced local immunity in the respiratory tract , including the number of pulmonary alveolar macrophages and lesions in the nasal mucosa , trachea , and lungs . 
 meanwhile , chs retarded dendritic cells ( dcs ) maturation and reduced the mrna levels of il-6 and ifn- significantly ( p < .05 ) . after the chs treatment 
 , mice were infected with h5n1 virus . 
 the mortality rate and viral load in the lungs of chs group were higher than those of tn group . 
 the results suggest that the chs treatment could suppress local immunity in the respiratory tract and innate host immunity in mice significantly and moderately increased the virulence in h5n1-infected mice .