Original Article Altered cytokine levels in bronchoalveolar lavage fluids from patients with mycoplasma pneumonia infection

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1 Int J Clin Exp Med 2016;9(8): /ISSN: /IJCEM Originl Article Altered cytokine levels in broncholveolr lvge fluids from ptients with mycoplsm pneumoni infection Yinle Ln, Zhimin Chen, Dehu Yng, Xi Wng, Yingshuo Wng, Yingchun Xu, Lnfng Tng Deprtment of Respirtion, Children s Hospitl, Medicl College of Zhejing University, Hngzhou , Chin Received December 9, 2015; Accepted July 9, 2016; Epub August 15, 2016; Published August 30, 2016 Abstrct: Mny reports hve demonstrted tht Mycoplsm pneumoni (M. pneumonie) infection my cuse the relese of multiple cytokines into sputum or serum smples. However, studies focusing on broncholveolr lvge fluid (BALF) smples re scrce. The im of this study ws to identify the cytokines ssocited with M. pneumonie infection, nd explore the possible fctors ffecting their secretion during cute lower respirtory trct infection. 60 children with confirmed M. pneumonie infection complicted with telectsis, ccording to chest X-ry or CT scn dt, nd 20 children with confirmed foreign bodies in the irwys (control group) were enrolled. Broncholveolr lvge (BAL) smples were obtined by flexible bronchoscopy, nd ssessed for the presence of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, tumor necrosis fctor (TNF)-α, nd interferon (IFN)-γ. Interestingly, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, nd IFN-γ mounts were incresed in M. pneumoni infected children compred with the control group. Multiple univrite nlyses of vrince nd multiple liner regressions showed tht fever during bronchoscopy mrkedly ffected IL-1β, IL-4, IL-10 nd IFN-γ concentrtions. In conclusion, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 nd IFN-γ levels were incresed in BALF from ptients with M. pneumoni. Incresed levels of secreted IL-1β, IL-4, IL-10 nd IFN-γ were ssocited with fever in these ptients. Keywords: Mycoplsm pneumoni, broncholveolr lvge, children, cytokines, fever Introduction Mycoplsm pneumoni (M. pneumonie) is one of the most common pthogens tht cuse lower respirtory trct infections, especilly in preschool children. M. pneumonie infection is self-limited disese, but its symptoms vry in different ge groups [1]. Older children re generlly more severely ffected thn younger ones, in contrst to other respirtory pthogens like respirtory syncytil virus (RSV) nd Streptococcus pneumoni [2]. Although irwy dmge plys n importnt role in M. pneumoni induced inflmmtion [3], n inpproprite immune response lso ccounts for pneumoni pthogenesis [1, 4]. Cytokines re intrcellulr signling proteins minly produced by immuno-competent cells. Their primry function is the regultion nd coordintion of immune responses [5]. Cytokine relese depends on specil mechnisms induced by the microorgnism itself [6, 7] or common rections like fever [8], during infection. The former provide some insights regrding the pthogen, while the ltter suggest which rections should be tken into considertion during investigtions iming to ssess how cytokine chnges my help understnd the pthogenesis of M. pneumonie infection. Although multiple studies hve reported IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, nd IFN-γ [5, 7, 9-11] chnges during M. pneumonie infection, consistent dt were not obtined [8, 12, 13]. Fever is the most common rection to infection. It cn be induced by multitude of substnces dministered peripherlly, rnging from inorgnic to orgnic compounds, or microbil nd mmmlin proteins; the existence of common endogenous fever meditor hs been postulted [5]. IL-1 receptor ntgonist (IL-1r), IL-1β, IL-6, nd IL-10 re considered endogenous pyrogens, while IL-1r nd IL-10 hve ntipyretic effects [5]. In ddition, fever limits the production of the fever-inducing IL-1β, nd influences the dptive immune response, fvoring Th2 cytokine production [8]. Therefore, fever is

2 n importnt rection tht should be tken into considertion in cytokine reserch in humns or niml models. Glucocorticoids (GCs) re gents tht downregulte the cell-medited immune response, which my be reflected by cytokine levels [6]. For mny severe clinicl M. pneumonie pneumoni cses tht cuse excessive immune response, GCs re effective in reducing the immune response nd voiding complictions [14, 15]. Like fever nd cytokines, GCs nd cytokines interct with ech other. GCs downregulte pro-inflmmtory cytokines while the ltter limit GC effects [16]. Therefore, tretment with GCs should lso be tken into ccount s it my ffect cytokine secretion. Refrctory M. pneumonie pneumoni (RMPP) is severe pneumoni chrcterizing cses with clinicl nd rdiologicl deteriortion despite pproprite ntibiotic therpy for 7 dys or more [17]. Its exct mechnism is uncler nd overrection of the immune response my ccount for this ilment, s mcrolides combined with corticosteroids re more effective thn mcrolides lone for tretment [17, 18]. Some cytokines [19] were even considered useful predictors of refrctory or severe Mycoplsm pneumonie pneumoni. Fiberoptic bronchoscopy (FOB) is n importnt technology for the exmintion nd tretment of lower respirtory trct disorders in children. As the lower respirtory trct is believed to be sterile nd close to lesions, detection of pthogens nd cytokines is more relible in BALF thn sputum, cerebrospinl fluid, or serum. Multiple studies hve reported vrying cytokine secretion levels in M. pneumonie infection, while ssessing serum or cerebrospinl fluid smples [9]. However, such studies focusing on broncholveolr lvge fluid (BALF) specimens re scrce, lthough BALF is close to lesions, since FOB is invsive nd only pplied to tret telectsis or foreign bodies. Although niml models re helpful in reducing smpling error, mny symptoms nd tretment outcomes will not be reflected in them, which contribute to disprte conclusions mong studies. Therefore, ppropritely defined conditions re meningful in this investigtion. We selected BALF s test smple close to lesions, in ptients complicted with telectsis, n indiction for broncholveolr lvge (BAL). We lso took into considertion the three most importnt conditions (fever, glucocorticoids tretment, nd RMPP), ccording to previous reports. Methods Study design Between Februry 2012 nd Februry 2013, children were eligible for enrollment if dignosed with M. pneumonie pneumoni, complicted with telectsis (X-ry or CT scn dt), or confirmed presence of foreign bodies. Finlly, 60 ptients with cliniclly confirmed M. pneumonie pneumoni complicted with telectsis nd 20 with foreign bodies were recruited t Children s Hospitl, Zhejing University School of Medicine, Hngzhou, Chin. No ptients hd severe underlying diseses such s bronchil sthm, chronic bronchitis, nd nsosinusitis, which might ffect the clinicl course. Nsophryngel swbs nd blood smples were obtined from ll ptients upon dmission. Then, the swbs were sent for sputum culture nd M. pneumoni detection by rel time PCR. The presence of respirtory syncytil virus (RSV), humn metpneumovirus (HMPV), influenz virus types A nd B, prinfluenz virus types 1-3, nd denovirus ws ssessed by direct immunofluorescence. Serum smples were sent for blood culture (BioMerieux, Frnce). All 80 ptients underwent BAL 3 dys fter dmission. IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α, nd IFN-γ levels in BALF smples were determined by sndwich enzyme linked immunosorbent ssy (ELISA) with specific kits (Diclone, Besncon Cedex, Frnce) ccording to the mnufcturer s instructions. The limits of detection were 6.5, 7.0, 0.7, 2.0, 29.0, 5.0, 20.0, 8.0, nd 5.0 pg/ml for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α, nd IFN-γ, respectively. BALF smples were lso sent for the ssessment of M. pneumonie lod. Clinicl prmeters (ge, gender, nd clinicl mnifesttions such s fever stte during BAL, GC tretment before BAL, nd fever durtion), lbortory exmintions (routine blood test, C-rection protein levels, lctte dehydrogense [LDH] mounts, nd type B ultrsound for pleurl effusion if needed), other complictions (rsh, cephlomeningitis, nd thrombotic thrombocytopenic purpur), nd prognosis dt (relief durtions of fever, cough, nd chest r Int J Clin Exp Med 2016;9(8):

3 Tble 1. Demogrphic dt nd cytokines of M. pneumonie pneumoni nd foreign body ptients Vribles M. pneumonie Pneumoni (n=60) Foreign Body (n=20) p-vlue Age, yers 5.23± ±0.54 <0.001 Mle 36 (60%) 16 (80%) Length of sty, dys 9 (7-12) 3 (2-4.75) <0.001 IL-1β (pg/ml) ( ) 7.05 ( ) <0.001 IL-2 (pg/ml) 9.54± ± IL-4 (pg/ml) 6.15 ( ) 0.03 (0-0.17) <0.001 IL-6 (pg/ml) ( ) 2.00 ( ) <0.001 IL-8 (pg/ml) ± ± <0.001 IL-10 (pg/ml) 7.23 ( ) 3.10 ( ) <0.001 IL-12 (pg/ml) ( ) ( ) TNF-α (pg/ml) ( ) ( ) IFN-γ (pg/ml) ( ) 2.12 ( ) <0.001 Results re presented s the men ± stndrd devition, number (percentge), or medin (25th-75th percentile). used to compre vrious groups, nd outcomes were presented s men ± stndrd devition (SD) for normlly distributed dt. For non-normlly distributed dt, the Mnn- Whitney U test ws used for comprison, nd outcomes were presented s medin with interqurtile rnge (25 th - 75 th ). Multiple univrite AN- OVA nd multiple liner regressions were used to nlyze fever, GC tretment, nd RMPP for their correltions with cytokines. P<0.05 ws considered sttisticlly significnt. Results diogrph bnormlities) were collected. Informed consent ws obtined from the prents of ll ptients, nd the study protocol ws pproved by the Ethics Committee of Zhejing University School of Medicine. Criteri for the determintion of M. pneumonie infection, fever, GC tretment, nd foreign body presence M. pneumonie infection ws chrcterized by: (i) detection of M. pneumonie DNA in the broncholveolr lvge fluid (BALF) smple by PCR; (ii) positive serologicl results, tht is, IgG titer >1:320 nd IgM titer >1.1 in the sme serum smple, detected with commercilly vilble ELISA kit (EUROIMMUN, Germny), ccording to the mnufcturer s instructions. A febrile stte ws defined s body temperture exceeding 37.5 C [20]. GC tretment ws considered fter ptients hd received three-dy methylprednisolone t 2 mg/kg/d intrvenously within 7 dys of BAL [15]. RMPP indicted cses with clinicl nd rdiologicl deteriortion despite pproprite ntibiotic therpy for 7 dys or more [17]. Fever durtion ws recorded from the first febrile dy fter onset. Foreign bodies were identified bsed on clinicl history, endoscopic inspection, nd, occsionlly, histopthologicl exmintion [21]. Sttisticl nlysis Independent-smples t test nd one-wy nlysis of vrince (ANOVA) with post hoc LSD were Demogrphic dt nd cytokine levels in ptients with M. pneumonie pneumoni nd foreign bodies Of the 80 children with M. pneumonie pneumoni complicted with telectsis or foreign bodies, 52 (65.0%) were mle. Men ptient ge ws 4.37 yers, rnging from 0.87 to 11.5 yers. M. pneumonie infection ws confirmed by serology nd rel-time PCR in serum nd BALF smples. No child required dmission to the intensive cre unit during hospitl sty, nd ll ptients were treted with mcrolides beginning t the outptient clinic or fter dmission. Regrding the demogrphics nd cytokine levels in ptients with M. pneumonie pneumoni nd foreign bodies, children with M. pneumonie pneumoni were older thn those with foreign bodies but no difference in gender distribution ws found between the two groups. Regrdless of conditions, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 nd IFN-γ levels were higher in the M. pneumonie pneumoni group compred with ptients with foreign bodies (Tble 1). Fever, RMPP, nd GC tretment in M. pneumonie pneumoni ptients Multi-fctor nlysis of vrince of fever, RMPP, nd GC tretment showed tht only febrile stte during FOB ws correlted with IL-1β, IL-4, IL-10, nd IFN-γ. RMPP nd GC tretment or combintion between the three prmeters did not show significnt correltions (Tble 2) Int J Clin Exp Med 2016;9(8):

4 Tble 2. Multiple fctor nlysis of vrince of fever, GCs tretment nd RMPP Cytokines/Fctors Fever RMPP GCs Fever+RMPP Fever+GCs RMPP+GCs IL-1β (pg/ml) IL-2 (pg/ml) IL-4 (pg/ml) IL-6 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-12 (pg/ml) TNF-α (pg/ml) IFN-γ (pg/ml) Results re presented s the p-vlue of ech fctor nd combintion in univrite multiple fctor nlysis of vrince. Tble 3. Multiple liner regressions of fever, RMPP nd GCs tretment Cytokines/Fctors R Regression Sig Fever RMPP GCs IL-1β (pg/ml) IL-2 (pg/ml) IL-4 (pg/ml) IL-6 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-12 (pg/ml) TNF-α (pg/ml) IFN-γ (pg/ml) Results re presented s the p-vlue of the coefficient of ech fctor in multiple liner regressions. Multiple liner regression nlysis of fever, RMPP, nd GC tretment showed tht only febrile stte during FOB ws significntly correlted with IL-1β, IL-4, nd IFN-γ levels. IL-2 nd IL-10 were relted to fever but the ssocitions were not significnt (P=0.059 nd P=0.110, respectively). RMPP nd GC tretment showed no significnt ssocitions with cytokine levels (Tble 3). Clinicl profiles nd cytokine levels in vrious groups Clinicl profiles nd cytokine levels in ptients with fever, no-fever, nd foreign bodies: A totl of 60 ptients with M. pneumonie pneumoni were subdivided into fever nd no-fever groups. The fever group showed longer fever durtion, fever durtion fter mcrolides, nd higher C-rective protein levels. As for cytokine mounts, the fever group showed significntly higher IL-2, IL-4, IL-10, nd IFN-γ levels; IL-1β ws lso higher but did not rech significnce Clinicl profiles nd cytokine levels in the RMPP, no-rmpp nd foreign body groups: A totl of 60 ptients with M. pneumonie pneumoni were divided into RMPP nd no-rmpp groups. Interestingly, the RMPP group showed longer fever durtion nd fever durtion fter mcrolides. Regrding cytokines, RMPP ptients showed no sttisticlly significnt difference compred with the no-rmpp group except for IFN-γ (Tble 5). Clinicl profiles nd cytokines in the GCs, no- GCs, nd foreign body groups: The 60 ptients with M. pneumonie pneumoni were subdivided into GC nd no-gc tretment groups. For clinicl profiles, the GC group showed longer durtion of fever nd fever durtion fter mcrolides. As for cytokines, the GC nd no-gc groups showed similr vlues (Tble 6). Discussion level compred with the no-fever group. In turn, the no-fever group showed higher levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, nd IFN-γ, compred with the foreign body group (Tble 4). The present study imed to nlyze the potentil effects of three conditions (fever, GC tretment, nd RMPP) on cytokine secretion into BALF ccording to previous reports, ssessing 9 cytokines in ptients with M. pneumonie pneumoni nd foreign bodies, respectively. Interestingly, we found tht fever ws more powerful thn the other two conditions, especilly in ltering IL-1β, IL-4, IL-10, nd IFN-γ secretion Int J Clin Exp Med 2016;9(8):

5 Tble 4. Clinicl profiles nd cytokines of fever, no fever nd foreign body groups Vribles Fever (n=30) No Fever (n=30) Foreign Body (n=20) p-vlue (Fever vs No Fever) p-vlue (Fever vs Foreign Body) p-vlue (No Fever vs Foreign Body) Age, yers 5.37± ± ± <0.001 <0.001 Mle 14 (46.7%) 22 (73.3%) 16 (80%) Length of sty, dys 10.93± ± (2-4.75) <0.001 <0.001 Fever durtion, dys 14.37± ± Fever durtion fter mcrolides, dys 5.72± ± Durtion of cough, dys 7.00 ( ) 8.00 ( ) Peripherl leukocyte count, 10 9 /l 8.55± ± C-rective protein, mg/l 61.79± ±22.75 <0.001 Lctte dehydrogense, IU/l ± ± IL-1β (pg/ml) ( ) ( ) 7.05 ( ) <0.001 <0.001 IL-2 (pg/ml) 9.84± ± ± IL-4 (pg/ml) ( ) 0.43 (0.04,7.23) 0.03 (0-0.17) < IL-6 (pg/ml) 51.86± ± ( ) IL-8 (pg/ml) ± ± ± <0.001 <0.001 IL-10 (pg/ml) 9.32 ( ) 5.12 ( ) 3.10 ( ) IL-12 (pg/ml) ( ) ( ) ( ) TNFα (pg/ml) ( ) ( ) ( ) IFN-γ (pg/ml) ( ) 4.43 ( ) 2.12 ( ) <0.001 < Results re presented s the men ± stndrd devition, number (percentge), or medin (25th-75th percentile) Int J Clin Exp Med 2016;9(8):

6 Tble 5. Clinicl profiles nd cytokines of RMPP, no RMPP nd foreign body groups Vribles RMPP (n=38) NRMPP (n=22) Foreign Body (n=20) p-vlue (RMPP vs NRMPP) p-vlue (RMPP vs Foreign Body) p-vlue (NRMPP vs Foreign Body) Age, yers 5.18± ± ± <0.001 <0.001 Mle 22 (57.9%) 14 (63.6%) 16 (80%) Length of sty, dys 11.13± ± (2-4.75) <0.001 <0.001 Fever durtion, dys 14.82± ±2.53 <0.001 Fever durtion fter mcrolides, dys 6.00 ( ) 3.00 ( ) Durtion of cough, dys 8.00 ( ) 6.5 ( ) Peripherl leukocyte count, 10 9 /l 8.36± ± C-rective protein, mg/l 49.03± ± Lctte dehydrogense, IU/l ± ± IL-1β (pg/ml) ( ) ( ) 7.05 ( ) < IL-2 (pg/ml) 9.66± ± ± IL-4 (pg/ml) 8.92± ± (0-0.17) < IL-6 (pg/ml) ( ) ( ) 2.00 ( ) IL-8 (pg/ml) ± ± ± <0.001 <0.001 IL-10 (pg/ml) 7.86 ( ) 5.06 ( ) 3.10 ( ) IL-12 (pg/ml) ( ) ( ) ( ) TNFα (pg/ml) 9.73 ( ) ( ) ( ) IFN-γ (pg/ml) 18.64( ) 5.96 ( ) 2.12 ( ) Results re presented s the men ± stndrd devition, number (percentge), or medin (25th-75th percentile) Int J Clin Exp Med 2016;9(8):

7 Tble 6. Clinicl profiles nd cytokines of GCs, no GCs nd foreign body groups Vribles GCs (n=22) No GCs (n=38) Foreign Body (n=20) p-vlue (GCs vs No GCs) p-vlue (GCs vs Foreign Body) p-vlue (No GCs vs Foreign Body) Age, yers 4.62± ± ± <0.001 <0.001 Mle 11 (50.0%) 25 (65.8%) 16 (80%) Length of sty, dys 11.68± ± (2-4.75) <0.001 <0.001 Fever durtion, dys 14.73± ± Fever durtion fter mcrolides, dys 6.27± ± Durtion of cough, dys 9.50 ( ) 7.00 ( ) Peripherl leukocyte count, 10 9 /l 9.96± ± C-rective protein, mg/l 42.49± ± Lctte dehydrogense, IU/l ± ± IL-1β (pg/ml) ( ) ( ) 7.05 ( ) IL-2 (pg/ml) 9.59± ± ± IL-4 (pg/ml) 7.29± ± (0-0.17) <0.001 IL-6 (pg/ml) 41.66± ± ( ) IL-8 (pg/ml) ± ± ± <0.001 <0.001 IL-10 (pg/ml) 8.30 ( ) 6.85 ( ) 3.10 ( ) IL-12 (pg/ml) ( ) ( ) ( ) TNFα (pg/ml) 5.22 ( ) ( ) ( ) IFN-γ (pg/ml) ( ) 9.89 ( ) 2.12 ( ) Results re presented s the men ± stndrd devition, number (percentge), or medin (25th-75th percentile) Int J Clin Exp Med 2016;9(8):

8 Fever is the multiphsic response of elevtion nd decline of the body core temperture, regulted by centrl thermoregultory mechnisms loclized in the preoptic re of the hypothlmus. The proinflmmtory cytokines IL-1, IL-6, nd TNF-α s well s nti-inflmmtory cytokines interleukin 1 receptor ntgonist (IL-1r) nd IL-10 hve been widely investigted for their pyrogenic or ntipyretic ctivities [5]. IL-1β is member of the IL-1 fmily, nd prototypic multifunctionl cytokine present in lmost every cell type [7]. IL-1β nd IL-10 levels increse in the febrile stte, nd ccount for their pyrogenic effects. IL-4 nd IFN-γ re representtive fctors for T helper type 2 (TH2) nd T helper type 1 (TH1) immune responses, respectively. High levels of IL-4 nd IFN-γ indicte intense inflmmtory nd nti-inflmmtory rections, which my be reflected by persistent fever, s IL-4 nd IFN-γ showed higher levels in the fever group compred with the nofever group. IL-6, representtive proinflmmtory cytokine nd mjor endogenous pyrogen [22], did not significntly increse in the febrile stte, s shown bove. A possible explntion is tht fever limits the production of fever-inducing proinflmmtory cytokines [8]. Incresed IL-1β but not elevted IL-6 levels during fever my be ttributed to different cytokine responses t vrious phses of fever, nd the temperture level might be involved s well. GCs re widely used in multiple severe infectious pneumoni, shortening fever durtion, reducing complictions, nd down-regulting the cell-medited immune response [15, 23, 24]. In this study, however, GC tretment did not lter cytokine profiles in ptients with M. pneumonie pneumoni. Although Remmelts HH [6] nd Tmur A [17] reported n obvious improvement in clinicl outcome fter dministrtion of dexmethsone or methylprednisolone, their use t high dosge hs not been proven sfe enough, nd our routine dose for severe infection is only 1-2 mg/kg/dy. Low dosge GC tretment hs distinct effects on different ptients [18], nd some my need high dosge [25]. Therefore, individul differences in both GC dosge nd sensitivity my be importnt [26]. RMPP is severe stte in M. pneumonie infection, nd Wng M nd collegues [27] reported tht serum TNF-α nd IFN-γ levels re higher in the RMPP group compred with nonrefrctory M. pneumonie pneumoni (NRMPP) ptients. Since fever is vitl to cytokine secretion in BALF s mentioned bove, it my lso ffect cytokine distribution in serum or other body fluids. Previous studies did not clssify ptients ccording to fever stte, nd most of them my hve been crried out with fever persistence, s RMPP is usully defined by long fever durtion. Unlike our outcome in BALF, different smple types nd other unblnced conditions like fever my ffect the dt. We lso found higher IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 nd IFN-γ levels in M. pneumoni infected children without fever compred with the 20 children confirmed with foreign bodies. These dt indicte tht M. pneumonie itself nd other unknown fctors cused the cytokine chnges observed in BALF smples. In ddition, significntly higher IL-4/IFN-γ rtios ( [ ]) were found in ptients with M. pneumonie pneumoni compred with the foreign body group ( [ ]). The cytokine profiles described bove suggest tht M. pneumonie infection cuses n unblnced Th1/Th2 immune response, which my contribute to llergic disorders, s reported by Koh YY nd collegues [28]. They my lso help distinguish M. pneumonie pneumoni from similr diseses such s pneumococcl pneumoni. Age is n importnt prmeter shown bove to be significntly different between ptients with M. pneumonie pneumoni nd the foreign body group. Foreign bodies nd telectsis re two mjor indictions of FOB. Most foreign bodies re found in toddlers, while telectsis cused by M. pneumonie pneumoni often ffects preschool children. However, there is not enough evidence to confirm the correltion between ge nd cytokine levels. We lso divided the 20 foreign body group into two subgroups, including the young (10 youngest children) nd old (10 oldest ones) subgroups, nd no significnt differences were found in cytokine levels between these two groups. These findings suggest tht there is no correltion between ge nd cytokine levels in BALF. A limittion of this study is tht pneumoni complicted with telectsis is not representtive of M. pneumonie infection s whole Int J Clin Exp Med 2016;9(8):

9 However, it is common mnifesttion, nd required for BAL; in ddition, it cn be considered severe M. pneumonie pneumoni to some extent. The reltively smll smple size lso limited the experimentl conditions to be considered. In conclusion, fever ffects the secretion into BALF of cytokines, especilly IL-1β, IL-4, IL-10, nd IFN-γ. M. pneumonie pneumoni cuses incresed IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, nd IFN-γ levels in BALF despite febrile stte. The exct mechnism needs to be further investigted. Disclosure of conflict of interest None. Address correspondence to: Zhimin Chen, Deprtment of Respirtion, Children s Hospitl, Medicl College of Zhejing University, Hngzhou , Chin. Tel: ; Fx: ; E-mil: lyl_lhb@163.com References [1] Wites KB nd Tlkington DF. Mycoplsm pneumonie nd its role s humn pthogen. Clin Microbiol Rev 2004; 17: , tble of contents. [2] Wites KB. New concepts of Mycoplsm pneumonie infections in children. Peditr Pulmonol 2003; 36: [3] Shimizu T, Kid Y nd Kuwno K. Cytodherence-dependent induction of inflmmtory responses by Mycoplsm pneumonie. Immunology 2011; 133: [4] Nrit M. Pthogenesis of extrpulmonry mnifesttions of Mycoplsm pneumonie infection with specil reference to pneumoni. J Infect Chemother 2010; 16: [5] Conti B, Tbren I, Andrei C nd Brtfi T. Cytokines nd fever. Front Biosci 2004; 9: [6] Remmelts HH, Meijvis SC, Biesm DH, vn Velzen-Bld H, Voorn GP, Grutters JC, Bos WJ nd Rijkers GT. Dexmethsone downregultes the systemic cytokine response in ptients with community-cquired pneumoni. Clin Vccine Immunol 2012; 19: [7] Yng J, Hooper WC, Phillips DJ nd Tlkington DF. Regultion of proinflmmtory cytokines in humn lung epithelil cells infected with Mycoplsm pneumonie. Infect Immun 2002; 70: [8] Boneberg EM nd Hrtung T. Febrile tempertures ttenute IL-1 bet relese by inhibiting proteolytic processing of the proform nd influence Th1/Th2 blnce by fvoring Th2 cytokines. J Immunol 2003; 171: [9] Nrit M, Tnk H, Togshi T nd Abe S. Cytokines involved in CNS mnifesttions cused by Mycoplsm pneumonie. Peditr Neurol 2005; 33: [10] Hoek KL, Duffy LB, Cssell GH, Di Y nd Atkinson TP. A role for the Mycoplsm pneumonie dhesin P1 in interleukin (IL)-4 synthesis nd relese from rodent mst cells. Microb Pthog 2005; 39: [11] Hrdy RD, Jfri HS, Olsen K, Wordemnn M, Htfield J, Rogers BB, Ptel P, Duffy L, Cssell G, McCrcken GH nd Rmilo O. Elevted cytokine nd chemokine levels nd prolonged pulmonry irflow resistnce in murine Mycoplsm pneumonie pneumoni model: microbiologic, histologic, immunologic, nd respirtory plethysmogrphic profile. Infect Immun 2001; 69: [12] Tnk H, Nrit M, Termoto S, Siki T, Oshi K, Igrshi T nd Abe S. Role of interleukin-18 nd T-helper type 1 cytokines in the development of Mycoplsm pneumonie pneumoni in dults. Chest 2002; 121: [13] Esposito S, Droghetti R, Bosis S, Clut L, Mrchisio P nd Principi N. Cytokine secretion in children with cute Mycoplsm pneumonie infection nd wheeze. Peditr Pulmonol 2002; 34: [14] Rdisic M, Torn A, Gutierrez P, Defrnchi HA nd Prdo P. Severe cute lung injury cused by Mycoplsm pneumonie: potentil role for steroid pulses in tretment. Clin Infect Dis 2000; 31: [15] Lee KY, Lee HS, Hong JH, Lee MH, Lee JS, Burgner D nd Lee BC. Role of prednisolone tretment in severe Mycoplsm pneumonie pneumoni in children. Peditr Pulmonol 2006; 41: [16] Dejger L, Vndevyver S, Pett I nd Libert C. Dominnce of the strongest: inflmmtory cytokines versus glucocorticoids. Cytokine Growth Fctor Rev 2014; 25: [17] Tmur A, Mtsubr K, Tnk T, Nigmi H, Yur K nd Fuky T. Methylprednisolone pulse therpy for refrctory Mycoplsm pneumonie pneumoni in children. J Infect 2008; 57: [18] Lu A, Wng L, Zhng X nd Zhng M. Combined tretment for child refrctory Mycoplsm pneumonie pneumoni with ciprofloxcin nd glucocorticoid. Peditr Pulmonol 2011; 46: [19] Miyshit N, Kwi Y, Inmur N, Tnk T, Akike H, Ternishi H, Wkbyshi T, Nkno T, Ouchi K nd Okimoto N. Setting stndrd for the initition of steroid therpy in refrctory Int J Clin Exp Med 2016;9(8):

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