Neonatal Streptococcus pneumoniae Pneumonia Induces an ...downloads.hindawi.com/journals/bmri/2019/1948519.pdf · ResearchArticle Neonatal Streptococcus pneumoniae Pneumonia Induces

Research ArticleNeonatal Streptococcus pneumoniae PneumoniaInduces an Aberrant Airway Smooth Muscle Phenotypeand AHR in Mice Model

Xin Peng12 Yi Wu12 Xiao Kong12 Yunxiu Chen12 Yonglu Tian12 Qinyuan Li12

Xiaoyin Tian3 Guangli Zhang3 Luo Ren2 and Zhengxiu Luo 3

1Key Laboratory of Pediatrics in Chongqing China2Department of Childrenrsquos Hospital of Chongqing Medical University of EducationKey Laboratory of Child Development and Disorders China3Department of Respiratory Medicine Childrenrsquos Hospital of Chongqing Medical University Chongqing China

Correspondence should be addressed to Zhengxiu Luo luozhengxiu816163com

Received 28 August 2018 Revised 12 November 2018 Accepted 23 December 2018 Published 6 January 2019

Academic Editor Enrico Heffler

Copyright copy 2019 Xin Peng et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Our previous study showed that neonatal S pneumoniae infection aggravated airway inflammation and airway hyperresponsiveness(AHR) in an OVA-induced allergic asthma model As airway smooth muscle (ASM) plays a pivotal role in AHR developmentwe aim to investigate the effects of neonatal S pneumoniae pneumonia on ASM structure and AHR development Non-lethalneonatal pneumonia was established by intranasally infecting 1-week-old BALBC mice with the S pneumoniae strain D39 Fiveweeks after infection the lungs were collected to assess the levels of 120572-SMA and the contractile proteins of ASM Our resultsindicate that neonatal S pneumoniae pneumonia significantly increased adulthood lung 120572-SMA and SMMHC proteins productionand aggravated airway inflammatory cells infiltration and cytokines release In addition the neonatal S pneumoniae pneumoniagroup had significantly higher Penh values compared to the uninfected controls These data suggest that neonatal S pneumoniaepneumonia promoted an aberrant ASM phenotype and AHR development in mice model

1 Introduction

Pneumonia is a common disease afflicting children espe-cially in developing countries [1] Streptococcus pneumoniae(S pneumoniae) is the predominant bacteria responsible forcommunity acquired pneumonia [2] Epidemiological stud-ies show that colonization of the neonatal respiratory tractwith S pneumoniae Haemophilus influenzae or Moraxellacatarrhalis significantly increases the risk of asthma in thefirst 5 years of life [3 4] Asthma characterized by chronicairway inflammation airway hyperresponsiveness (AHR)and remodeling is one of the most common chronic airwaydiseases among children worldwide [5 6] AHR which islargely attributed by airway structural changes includingairway smooth muscle (ASM) hypertrophyhyperplasia haslong been considered a cardinal feature of asthma [7 8]Changes in structure andor function of ASM have been

observed in both asthma patients and experimental asthmamodels [9 10] Since ASM is responsible for airway con-striction [11] alterations including hyperplasiahypertrophyandor dysregulation of contractile proteins of ASM cantrigger AHR and airway remodeling [12 13] Our previousstudy stated neonatal S pneumoniae infection aggravatesairway inflammation and AHR in the ovalbumin (OVA) -induced allergic asthma model [14] Whether neonatal Spneumoniae infection induces asthma is associated withthe alterations of ASM structure andor function remainsunclear This intriguing observation promoted us to furtherinvestigate the effects of neonatal S pneumoniae pneumoniaon adulthood ASM structure and AHR development absentfrom allergen challenge In this study we found that neonatalS pneumoniae pneumonia mice had significantly higherlevels of alpha-smooth-muscle-actin (120572-SMA) and smooth-muscle-myosin-heavy-chain (SMMHC) in adulthood lung

HindawiBioMed Research InternationalVolume 2019 Article ID 1948519 8 pageshttpsdoiorg10115520191948519

2 BioMed Research International

tissues as revealed by immunohistochemistry and Westernblot analysis The expression of smooth muscle-specific genes(actin-1205722-smooth-muscle-aorta Acta2 and myosin-heavy-chain-11-smooth-muscle Myh11 respectively) was remark-ably increased in neonatal S pneumoniae pneumonia groupconsistent with the Western blotting and histologic resultsFurthermore AHRwas also remarkably higher in the S pneu-moniae pneumonia group compared to the controls Takentogether neonatal S pneumoniae pneumonia promoted anaberrantASMphenotype andAHRdevelopment absent fromallergen challenge in mice model

2 Materials and Methods

21 Establishment of the Non-Lethal Neonatal PneumoniaModel Using S pneumoniae All experimental protocols wereapproved by the Institutional Animal Care and ResearchAdvisory Committee of the Chongqing Medical UniversityThe animals were treated in accordance with the guidelinesissued by the Chinese Council on Animal Care ParturientBALBC mice were purchased from Animal Resources Cen-tre of Chongqing medical university housed separately andclosely monitored for births Newborn mice were housed at25∘Cunder a 12 h lightdark cycle and providedwith adequatefood and waterWe established a non-lethal neonatal S pneu-moniae model of pneumonia in these neonates as describedin our previous study [14] Briefly S pneumoniae D39 strainwas inoculated into tryptic soy broth (Pangtong China) andcultured for 12-14h at 37∘C under 5 CO

2 Neonatal (1-

week-old) BALBC mice were infected intra-nasally with 2times 106 colony-forming units (CFU) of S pneumonia in 5120583lphosphate buffered saline (PBS) while the mock-infectedcontrols received the same volume of sterile PBS Five weekspostinfection the lungswere removed and homogenized andthe tissue homogenates were cultured on blood agar for 24 h(at 37∘C under 5 CO

2) to determine the bacterial load

22 Histology of the Lungs Five weeks after pneumoniaie when the neonates had reached adulthood the micewere euthanized with a lethal dose of 10 chloralhydrate(03ml100g intraperitoneally) and their lungs were har-vested The left lungs were desiccated and embedded inparaffin after fixing in 4 paraformaldehyde Tissue sections(4120583m thick) were stained with hematoxylin and eosin (HampESigma-Aldrich) and the lung lesions were semiquantitativelyscored as described previously [15] 0 points for no cell 1 pointfor few cells 2 points for a ring of inflammatory cells 1 celllayer deep 3 points for a ring of inflammatory cells 2 to 4cells deep 4 points for a ring of inflammatory cells of gt4 cellsdeep

23 Bronchoalveolar Lavage Fluid (BALF) Analysis Follow-ing euthanization of the mice as described above the BALFwas obtained by washing the lungs five times with 1ml ice-cold PBS The number of total cells differential cell countsand the level of cytokines including IL-4 IL-5 IL-13 IL-17A and INF-120574were determined as previously described [14]The cytokine levels were determined using specific enzyme-linked immunosorbent assay (ELISA) kits (Neobioscience

Shenzhen China) according to the manufacturerrsquos instruc-tions

24 Immunohistochemistry (IHC) The lung tissue sectionswere deparaffinized and dehydrated as per standard pro-tocols After blocking endogenous peroxidase activity andnon-specific staining with H

2O2and 5 bovine serum

albumin (BSA) respectively the sections were incubatedwith mouse monoclonal anti-120572-SMA rabbit monoclonalanti-SMMHC or rabbit monoclonal anti-smooth muscle 22alpha (SM22120572) antibodies (all diluted 1100 Sigma-AldrichSt Louis MO) at 4∘C for 12 h The sections were washedwith PBS and incubated with secondary antibody for 30minat 37∘C The positive signals were developed using the 331015840-diaminobenzidine (DAB) chromogen and the sections werecounterstained with hematoxylin The basement membraneperimeter (Pbm) and the 120572-SMA+ SMMHC+ and SM22120572+areas [16] were outlined and analyzed with the Image-ProPlus 60 software (Image-Pro 60 USA) For accurate mor-phometric quantification Pbm2 was first used to standardizethe region of interest ie sections of the airway wall andconnective tissue attachments excluding the blood vessels[16ndash18] The 120572-SMA+ areaPbm2 SMMHC+ areaPbm2 andSM22120572+ areaPbm2 ratios were calculated to respectivelydefine the 120572-SMA+ area SMMHC+ area and SM22120572+ areaaround the airway [17 19]

25 Western Blotting Total protein was extracted from theharvested lungs of adult mice and denatured with theSDS-PAGE loading buffer Equal amounts of protein fromeach group were separated on 10 SDS-PAGE and thentransferred onto polyvinylidene difluoride (PVDF) mem-branes (Millipore) The membranes were incubated withmouse anti-120572-SMA (1500 Sigma) rabbit anti-SMMHC(11000 Sigma) rabbit anti-SM22120572 (11000 Sigma) or rab-bit anti-GAPDH (11000 Proteintech) antibody for 12 h at4∘C After washing with PBS the membranes were incu-bated with the secondary antibody and the positive bandswere quantified using Quantity-One software relative toGAPDH

26 Real-Time Quantitative PCR (RT-qPCR) Total RNAwasextracted from the lungs of adult mice with TRIzol (Invit-rogen CA) and reverse transcribed into cDNA using thePrimeScript RT kit (TaKaRa Japan) The relative expressionsof 120572-SMA gene (Acta2) SMMHC gene (Myh11) and SM22120572gene (transgelin Tagln) were detected using the RT-qPCRassay kit from Life Technologies The sequences of therespective forward (F) and reverse (R) primers are as follows

Acta2-F 51015840-TGCTGGACTCTGGAGATGGTGTG-31015840

Acta2-R 51015840-CGGCAGTAGTCACGAAGGAAT-AGC-31015840

Myh11-F 51015840-CCATTGCCGACACAGCCTACAG-31015840

Myh11-R 51015840-GGATGCCACCACAGCCAAGTAC-31015840

Tagln-F 51015840-AGATGGAACAGGTGGCTCAATTCT-TG-31015840

BioMed Research International 3

1 2 3 4 5 6 7 140

1000

2000

3000

4000

Post Pneumonia (Days)

pneu

mon

iae c

fum

lSt

rept

ococ

cus

in lu

ng h

omog

enat

e

(a)

1 2 3 4 5 6 7 14 21 28000

005

010

015

020

controlSpp

days post infection

Lung

wei

ght (

g)

lowastlowastlowast

lowastlowast

(b)

1 2 3 4 5 6 7 14 21 280

40

controlSpp

days post infection

Body

wei

ght (

g)

10

20

30

lowast lowast lowast

lowast lowastlowast lowastlowast

(c)

Figure 1 Neonatal S pneumoniae pneumonia affects lung bacterial load and lung and body weight (a) S pneumoniae colony counts inthe lung (b) Lung weight (c) Body weight lowastPlt005 lowastlowastPlt001 compared to the control group Spp S pneumoniae pneumonia

Tagln-R 51015840- CCTTCATAGAGGTCAACAGTC-TGGAAC-31015840

GAPDH-F 51015840-CAGCGACACCCACTCCTCCAC-CTT-31015840

GAPDH-R 51015840- CATGAGGTCCACCACCCTGTT-GCT -31015840

27 Airway Hyperresponsiveness (AHR) Five weeks afterpneumonia airway hyperresponsiveness (AHR) was deter-mined by whole-body plethysmograph (Emka instrumentFrance) as previously described [20 21] Briefly con-scious and spontaneously breathing mice were exposed toaerosolized normal saline followed by increasing concen-trations of aerosolized methacholine (3125 625 125 25and 50mgml Sigma USA) in normal saline for 3min Thehighest enhanced pauses (Penh) value obtained during eachmethacholine challenge was expressed as a proportion ofthe basal Penh value in response to normal saline challengePenh is a dimensionless value which reflects airway hyper-responsiveness It represents a function of the ratio of peakexpiratory flow to peak inspiratory flow and a function of thetiming of expiration

28 Statistical Analysis Data were expressed as means plusmn SDOne-way analysis of variance (ANOVA) and Studentrsquos t-testswere used to compare the groups All statistical analyses wereperformed using Graph Pad Prism (version 50 Graph PadLa Jolla CA USA) and plt005 was considered statisticallysignificant

3 Results

31 Duration and Severity of Neonatal Pneumonia Causedby S pneumoniae Infection To determine the duration andseverity of neonatal pneumonia the pulmonary bacterial loadand the lung and body weights of the mice were assessedS pneumoniae were cleared from the lungs within 7 days ofinfection While the lung and the total body weight increasedin the uninfected control mice with age the S pneumoniaeinfected mice lost a significant amount of body weight within

the first week post-infection although their lungs were heav-ier compared to the control group 3 to 6 days post-infection(Figures 1(a)ndash1(c)) After 7 days of initiating pneumonia thelung and body weights of the infected mice were restoredto that of the uninfected mice These results indicated thatneonatal S pneumoniae pneumonia had minimal effects onlung bacterial load and lung and body weight of adult mice

32 Neonatal S pneumoniae Pneumonia Altered AirwaySmooth Muscle Productions in Mice Model To determinethe effects of neonatal S pneumoniae pneumonia on airwaysmooth muscle productions we analyzed the expressionof 120572-SMA and the contractile proteins of ASM (SMMHCand SM22120572) in adulthood lung tissues Neonatal S pneu-moniae pneumonia significantly increased the relative 120572-SMA positive area (120572-SMA+ areaPbm2) (001071 plusmn 0003081vs 0005089 plusmn 0001586 Plt001 Figures 2(a) and 2(b))Acta2-mRNA (2995 plusmn 09433 vs 09065 plusmn 02148 Plt001Figure 2(c)) and 120572-SMA protein levels (1338 plusmn 05061 vs06376 plusmn 01443 Plt001 Figures 2(d) and 2(e)) in the adultlungs compared to the uninfected controls In additionthe relative SMMHC positive area (002875 plusmn 001251 vs0008826 plusmn 0003849 Plt005) Myh11-mRNA (2270plusmn 05375vs 1084 plusmn 03131 Plt001) and SMMHC protein levels (1291plusmn 04872 vs 07768 plusmn 02793 Plt001) were also significantlyhigher in the S pneumoniae pneumonia group comparedto the uninfected controls (Figures 2(f)ndash2(j)) In contrastthe relative SM22120572 positive area Tagln-mRNA and SM22120572protein levelswere similar in both groups (Figures 2(k)ndash2(o))These results indicated neonatal S pneumoniae pneumoniainduced an increase in airway smooth muscle mass andairway remodeling in mice model

33 Neonatal S pneumoniae Pneumonia Aggravated AirwayInflammatory Cells Infiltration and Cytokines Release in MiceModel Five weeks after pneumonia the lung tissues andBALF were collected to assess airway inflammation Theneonatal S pneumoniae pneumonia mice had significantlyhigher infiltration of inflammatory cells compared to thecontrols (Figure 3(a)) The inflammation scores for pul-monary peri-bronchiolitis (2584plusmn 02379 vs 08738plusmn 03544


Control Spp

-SMA

(a)

-S

MA

+are

aPb

m

Control Spp0000

0005

0010

0015 lowastlowast

(b)

control Spp0

1

2

3

4

5

lung

tiss

ueA

cta2

mRN

AG

APD

H in

lowastlowast

(c)

GAPDH

Control Spp

-SMA

(d)

Control Spp00

05

10

15

20

-S

MA

pro

tein

GA

PDH

lowastlowast

(e)

SMMHC

Control Spp

(f)

SMM

HC

+ar

eaPbm

Control Spp000

001

002

003

004

005lowast

(g)

Control Spp0

1

2

3

lung

tiss

ueM

yh11

mRN

AG

APD

H in

lowastlowast

(h)

SMMHC

GAPDH

Control Spp

(i)

Control Spp00

05

10

15

20

SMM

HC

prot

ein

GA

PDH

lowastlowast

(j)

Control Spp

SM22

(k)

SM22

+

area

Pbm

Control Spp0000

0005

0010

0015

0020

0025

(l)

Control Spp00

05

10

15

20

lung

tiss

ueTa

gln

mRN

AG

APD

H in

(m)

Control Spp

GAPDH

SM22

(n)

Control Spp00

05

10

15

20

SM22

p

rote

inG

APD

H

(o)

Figure 2 Neonatal S pneumoniae pneumonia alters airway smooth muscle productions in mice model IHC staining of lung tissuesfrom both mock-infected (control) and neonatal S pneumoniae pneumonia mice (Spp) showing the in situ expression of 120572-smooth muscleactin (120572-SMA) (a) smooth muscle myosin heavy chain (SMMHC) (f) and smooth muscle 22 alpha (SM22120572) (k) (400x magnification) Therelative 120572-SMA-positive (120572-SMA+ areaPbm2) (b) SMMHC-positive (SMMHC+ areaPbm2) (g) and SM22120572-positive (SM22120572+ areaPbm2)(l) areas are also shown RT-qPCR was used to analyze Acta2-mRNA (c) Myh11-mRNA (h) and Tagln-mRNA (m) levels in the lung tissuesThe 120572-SMA (d e) SMMHC (i j) and SM22120572 (n o) protein levels in the lung tissues were analyzed byWestern blotting All data are presentedas means plusmn SD (n =5group) lowastlowastPlt001 compared to the control group

HampE stain

Control Spp

(a)

Control Spp0

1

2

3

peri

-bro

nchi

ole i

nflam

mat

ion

His

tolo

gica

l sco

re o

f

lowastlowastlowast

(b)

Control Spp0

1

2

3

peri

-vas

cula

r infl

amm

atio

nH

istol

ogic

al sc

ore o

f lowastlowastlowast

(c)

Control Spp0

1

2

3

His

tolo

gica

l sco

re o

f alv

eola

rin

flam

mat

ion

lowastlowast

(d)

Figure 3 Neonatal S pneumoniae pneumonia increases adulthood inflammatory cell infiltration in the lung tissues HampE stainingof lung samples from mock-infected and neonatal S pneumoniae pneumonia mice 5 weeks after pneumonia (a) (200x magnification)Histological scores of pulmonary peri-bronchiolitis (b) pulmonary perivasculitis (c) and pulmonary alveolitis (d) Data are shown as meanplusmn SD (n=6-8 micegroup) lowastlowastPlt001 lowast lowast lowastPlt0001 compared to the control group


Table 1 Neonatal S pneumoniae pneumonia increases inflammatory cell accumulation in the BALF

Group n Total cells Neutrophils Macrophage Lymphocyte Eosinophils(times105) (times104) (times104) (times104) (times104)

Control 5 421 plusmn 0765 685 plusmn 104 322 plusmn 474 849 plusmn 259 0283 plusmn 00448

Spp 5 168 plusmn 536 lowast lowast 199 plusmn 517 lowast lowast 574 plusmn 418 lowast lowastlowast 335 plusmn 118 lowast lowastlowast 11 plusmn 0421 lowast lowast

Data are shown as mean plusmn SDlowastlowastPlt001 lowast lowast lowast Plt0001 compared to the control group

Table 2 Neonatal S pneumoniae pneumonia increases cytokines production in BALF (pgsdotmlminus1)

Group n IL-4 IL-5 IL-13 IL-17A INF-120574Control 6 205 plusmn 409 155 plusmn 704 124 plusmn 732 628 plusmn 1138 449 plusmn 809

Spp 6 291 plusmn 453lowast 285 plusmn 116 lowast lowast 217 plusmn 911lowast 1157 plusmn 1045 lowast lowastlowast 198 plusmn 637 lowast lowast

Data are shown as mean plusmn SDlowastPlt005 lowastlowastPlt001 lowast lowast lowast Plt0001 compared to the control group

Plt0001) pulmonary peri-vasculitis (2043 plusmn 03740 vs 02513plusmn 03468 Plt0001) and pulmonary alveolitis (1876 plusmn 08537vs 06650 plusmn 06412 Plt001) in the infected mice were remark-ably increased compared to the controls (Figures 3(b)ndash3(d))

Consistentwith the lung inflammation results neonatal Spneumoniae pneumonia mice had significantly higher countsof total inflammatory cells neutrophils macrophages lym-phocytes and eosinophils in the BALF compared to controls(Table 1) In addition the levels of IL-4 IL-5 IL-13 and IL-17A in BALF were also significantly higher in the pneumoniagroup (Table 2) Our results indicate neonatal S pneumoniaepneumonia aggravated adulthood airway inflammatory cellsinfiltration and cytokines release

34 Neonatal S pneumoniae Pneumonia Promoted AHR De-velopment in Mice Model Five weeks after neonatal S pneu-moniae pneumonia AHR was assessed by the calculation ofPenh values (ie enhanced respiratory pausing) No signifi-cant differences were found in the baseline of airway respon-siveness of both groups following normal saline challengeWith the increase of the concentration of methacholinethe airway responsiveness of the S pneumoniae pneumoniagroupwas significantly higher compared to the controlswhenexposed to 125mgml (2005 plusmn 06622 vs 04327 plusmn 003387Plt0001) 25mgml (2845 plusmn 06230 vs 04580 plusmn 002559Plt0001) and 500mgml (3335plusmn 06364 vs 04714plusmn 004637Plt0001) methacholine (Figure 4) Therefore neonatal Spneumoniae pneumonia promoted AHR development inmice model

4 Discussion

Neonatal S pneumoniae infection promotes experimentalasthma development in adults [14] While Preston et al [22]stated adulthood S pneumoniae infection protected againstallergic asthma in mice model Al-Garawi et al [23] showedthat neonatal exposure to allergens (HMD) in the presenceof acute influenza virus infection induced lung remodelingand imprinted an asthmatic phenotype in adult BALBCmice Horvat et al [24 25] found that neonatal and notadult respiratory Chlamydia pneumoniae infections altered

0 3125 625 125 25 500

1

2

3

4

5

ControlSpp

Penh

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 4 Neonatal S pneumoniae pneumonia promotes AHRdevelopment in adult mice Five weeks after pneumonia whole-body plethysmography was conducted in mock-infected (control)and neonatal S pneumoniae pneumonia mice (Spp) with metha-choline All data are presented as mean plusmn SD (n=6-8 micegroup)lowast lowast lowast Plt0001 compared to the control group

lung function and structure and enhanced the severity ofrespiratory allergies in later life in mice Whether neonatal Spneumoniae infection promotes allergic asthma by inducingairway remodeling and AHR in the absence of allergens is notcompletely clarified Herewe showed for the first time inmicemodel that neonatal S pneumoniae pneumonia increased theexpression of 120572-SMA in the airway partially altered the ASMphenotype and induced airway inflammation and AHR

ASM is the main structural component of the airwaystructure and controls the diameter of bronchi and bronchi-oles via contractile function [11] ASM remodeling in theform of ASM cells hypertrophy hyperplasia and changes incontractility is associated with AHR and airway remodeling[26] Viruses such as rhinovirus influenza and respiratorysyncytial virus can promote ASM hyperplasia and enhancethe contractility and secretory functions of ASM [27ndash29] Cpneumoniae has been shown to induce ASM proliferation in


vitro via the increased secretion of IL-6 PDGF and FGF [30]However few studies have investigated the effects of bacterialinfection on ASM

To the best of our knowledge this is the first studyto explore the effects of neonatal S pneumoniae infectionon the expression of airway 120572-SMA and ASM contractileproteins in adults We found that neonatal S pneumoniaepneumonia upregulated airway Acta2 and Myh11 mRNAsand 120572-SMA and SMMHC proteins in the adulthood miceand also promoted AHR Our results are consistent withprevious studies which correlated airway structural changestoAHRdevelopment [31ndash33]The remodeling and contractilecapacity of ASM can be enhanced by inflammatory mediatorsfurther to contribute to AHR [34] Our data showed thatneonatal S pneumoniae pneumonia can increase the infil-tration of inflammatory cells both in the lung tissues andin the BALF along with releasing higher levels of the pro-inflammatory cytokines (IL-4 IL-5 IL-13 and IL-17A) TypeII (Th2) cytokines including IL-4 IL-5 and IL-13 can pro-mote AHR airway remodeling and mucus hypersecretion[35] IL-4 enhances ASM contractility and proliferation ofthe ASM cells [36] IL-13 also promotes ASM contraction andremodeling and thus leads to AHR via calcium accumulation[37] Finally IL-17Anot only promotesASM cell proliferationand contraction but also induces the secretion of pro-AHRcytokines and chemokines [29 38] We found that neonatalS pneumoniaepneumonia significantly increased the levels ofAHR IL-4 IL-5 IL-13 and IL-17A relative to controls whichis consistent with the above studies Therefore in this studywe speculate that the secretion of inflammation cytokinesand chemokines may promote ASM remodeling and AHR ofadult mice The specific mechanism of action remains to befurther studied

As one of the contractile ASM proteins SMMHC expres-sion increased is highly involved in the enhancement ofairway remodeling which contributes to AHR during chronicasthma in mice model [39] Chronic inflammation can regu-late the expression of smoothmuscle-specific contractile pro-teins including SMMHC [40] while CD4+ T cells are knownto induce SMMHC and promote AHR [41] Consistent withthis neonatal S pneumoniae infection promotes CD4+ Tcells production in adulthood [14] We found that neonatal Spneumoniae infection significantly increased SMMHC levelswhichmay be an important factor for airway remodeling andAHR Growing evidence indicates that airway inflammationand ASM remodeling also synergize with each other [17 42]Airway inflammation induced by dysfunctional ASM canpromote SMMHC protein expressions which can furtheraggravate AHR by enhancing ASM remodeling [43]

Our study also had some limitations The invasive tech-nique has been used widely to measure airway resistanceand hyperresponsiveness Other studies and our laboratorystudies demonstrate that Penh can be used as an indicatorof AHR [15 44ndash50] Here we only used Penh to representairway responsiveness due to the high mortality with aninvasive technique On the other hand there were a series ofconnections between ASM and inflammation Some studiesindicated that ASM remodeling triggers airway inflammationand AHR by producing cytokines [51 52] Another study

showed that the cytokines chemokines and matrix proteinsproduced by the ASM cells were necessary for their in vitroproliferation [53] In addition airway inflammation triggeredby dysfunctional ASM can further promote its contraction[54] In this study we did not investigate the direct linkbetween ASM and inflammation further studies are neededto clarify the direct link between ASM remodeling andairway inflammation induced by neonatal S pneumoniaepneumonia

5 Conclusions

In conclusion neonatal S pneumoniae pneumonia promotesairway smooth muscle phenotype and AHR in adult micewhich provides a theoretical basis for asthma prevention

Abbreviations

Acta2 Actin-1205722-smooth-muscle-aortaAHR Airway hyperresponsivenessASM Airway smooth muscleBALF Bronchoalveolar lavage fluidMyh11 Myosin-heavy-chain-11-smooth-musclePBS Phosphate buffered salineS pneumoniae Streptococcus pneumoniaeSM22120572 Smooth-muscle-22-alphaSMMHC Smooth-muscle-myosin-heavy-chainTagln Transgelin120572-SMA Alpha smooth muscle actin

Data Availability

The datasets used andor analyzed during the current studyare available from the corresponding author upon reasonablerequest

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (81270086 and 814700222)the Science and Technology Department of Chongqing(cstc2017jcyjB0160) and the Science and Technology Depart-ment of Yuzhong District Chongqing (20170120)

References

[1] R Izadnegahdar A L Cohen K P Klugman and S A QazildquoChildhood pneumonia in developing countriesrdquo The LancetRespiratory Medicine vol 1 no 7 pp 574ndash584 2013

[2] I C Michelow K Olsen J Lozano et al ldquoEpidemiology andClinicalCharacteristics ofCommunity-AcquiredPneumonia inHospitalized Childrenrdquo Pediatrics vol 113 no 4 I pp 701ndash7072004


[3] N H Vissing B L K Chawes and H Bisgaard ldquoIncreased riskof pneumonia and bronchiolitis after bacterial colonization ofthe airways as neonatesrdquo American Journal of Respiratory andCritical Care Medicine vol 188 no 10 pp 1246ndash1252 2013

[4] M Korppi ldquoManagement of bacterial infections in childrenwith asthmardquo Expert Review of Anti-infective Therapy vol 7 no7 pp 869ndash877 2009

[5] T Kawayama T Kinoshita K Matsunaga et al ldquoRole ofRegulatory T cells in Airway Inflammation in Asthmardquo TheKurume Medical Journal vol 64 no 3 pp 45ndash55 2017

[6] W Eder M J Ege and E Von Mutius ldquoThe asthma epidemicrdquoThe New England Journal of Medicine vol 355 no 21 pp 2226ndash2235 2006

[7] W W Busse ldquoThe relationship of airway hyperresponsive-ness and airway inflammation airway hyperresponsiveness inasthma its measurement and clinical significancerdquo Chest vol138 no 2 pp 4Sndash10S 2010

[8] D G Chapman and C G Irvin ldquoMechanisms of airway hyper-responsiveness in asthma The past present and yet to comerdquoClinical ampExperimental Allergy vol 45 no 4 pp 706ndash719 2015

[9] G Lezmi P Gosset A Deschildre et al ldquoAirway remodelingin preschool children with severe recurrent wheezerdquo AmericanJournal of Respiratory and Critical CareMedicine vol 192 no 2pp 164ndash171 2015

[10] X Wen J Yan X-R Han et al ldquoPTEN gene silencingcontributes to airway remodeling and induces airway smoothmuscle cell proliferation in mice with allergic asthmardquo Journalof Thoracic Disease vol 10 no 1 pp 202ndash211 2018

[11] A J Halayko T Tran and R Gosens ldquoPhenotype and func-tional plasticity of airway smooth muscle Role of caveolae andcaveolinsrdquo Proceedings of the American Thoracic Society vol 5no 1 pp 80ndash88 2008

[12] Y S Prakash ldquoAirway smooth muscle in airway reactivityand remodeling what have we learnedrdquo American Journal ofPhysiology-Lung Cellular andMolecular Physiology vol 305 no12 pp L912ndashL933 2013

[13] Y S Prakash ldquoEmerging concepts in smooth muscle contribu-tions to airway structure and function implications for healthand diseaserdquo American Journal of Physiology-Lung Cellular andMolecular Physiology vol 311 no 6 pp L1113ndashL1140 2016

[14] B Yang R Liu T Yang et al ldquoNeonatal Streptococcus pneumo-niae infection may aggravate adulthood allergic airways diseasein association with IL-17Ardquo PLoS ONE vol 10 no 3 Article IDe0123010 2015

[15] N Zang J Zhuang Y Deng et al ldquoPulmonary C fibers modu-lateMMP-12 production via PAR2 and are involved in the long-term airway inflammation and airway hyperresponsivenessinduced by respiratory syncytial virus infectionrdquo Journal ofVirology vol 90 no 5 pp 2536ndash2543 2016

[16] A Bai D H Eidelman J C Hogg et al ldquoProposed nomencla-ture for quantifying subdivisions of the bronchial wallrdquo Journalof Applied Physiology vol 77 no 2 pp 1011ndash1014 1994

[17] C Y Lou M Li and L Li ldquoDynamic changes in percentagesof CD4+ CD25+ regulatory T cells and Th17 cells in process ofairway remodeling inmousemodel of asthmardquo Zhongguo DangDai Er Ke Za Zhi vol 17 pp 994ndash1000 2015

[18] I Labonte M Hassan P Risse et al ldquoThe effects of repeatedallergen challenge on airway smooth muscle structural andmolecular remodeling in a rat model of allergic asthmardquoAmerican Journal of Physiology-Lung Cellular and MolecularPhysiology vol 297 no 4 pp L698ndashL705 2009

[19] Y Wang C Xue F Dong et al ldquoHydroxysafflor yellow Aattenuates small airway remodeling in a rat model of chronicobstructive pulmonary diseaserdquo Biological amp PharmaceuticalBulletin vol 37 no 10 pp 1591ndash1598 2014

[20] N Zang X Xie Y Deng et al ldquoResveratrol-mediated gammainterferon reduction prevents airway inflammation and air-way hyperresponsiveness in respiratory syncytial virus-infectedimmunocompromisedmicerdquo Journal of Virology vol 85 no 24pp 13061ndash13068 2011

[21] L Zhang H Gao T Yang et al ldquoInfant 7-valent pneumococ-cal conjugate vaccine immunization alters young adulthoodCD4+T cell subsets in allergic airway disease mouse modelrdquoVaccine vol 32 no 18 pp 2079ndash2085 2014

[22] J A Preston A N Thorburn M R Starkey et al ldquoStreptococ-cus pneumoniae infection suppresses allergic airways disease byinducing regulatory T-cellsrdquo European Respiratory Journal vol37 no 1 pp 53ndash64 2011

[23] A A Al-Garawi R Fattouh T D Walker et al ldquoAcute but notresolved influenza a infection enhances susceptibility to housedust mite-induced allergic diseaserdquoThe Journal of Immunologyvol 182 no 5 pp 3095ndash3104 2009

[24] J C Horvat K W Beagley M A Wade et al ldquoNeonatalchlamydial infection induces mixed T-cell responses that driveallergic airway diseaserdquo American Journal of Respiratory andCritical Care Medicine vol 176 no 6 pp 556ndash564 2007

[25] J C Horvat M R Starkey R Y Kim et al ldquoEarly-lifechlamydial lung infection enhances allergic airways diseasethrough age-dependent differences in immunopathologyrdquo TheJournal of Allergy and Clinical Immunology vol 125 no 3 pp617ndash625 2010

[26] B G J Dekkers I S T Bos A J Halayko J Zaagsma andH Meurs ldquoThe laminin 1205731-competing peptide YIGSR inducesa hypercontractile hypoproliferative airway smooth musclephenotype in an animal model of allergic asthmardquo RespiratoryResearch vol 11 article no 170 2010

[27] B Yeganeh C Xia H Movassagh et al ldquoEmerging mediatorsof airway smooth muscle dysfunction in asthmardquo PulmonaryPharmacology ampTherapeutics vol 26 pp 105ndash111 2013

[28] A K Mehta T Doherty D Broide and M Croft ldquoTumornecrosis factor familymember LIGHTactswith IL-1120573andTGF-120573 to promote airway remodeling during rhinovirus infectionrdquoAllergy vol 73 pp 1415ndash1424 2018


[30] J Rodel M Woytas A Groh et al ldquoProduction of basicfibroblast growth factor and interleukin 6 by human smoothmuscle cells following infection with Chlamydia pneumoniaerdquoInfection and Immunity vol 68 no 6 pp 3635ndash3641 2000

[31] R M Pascual and S P Peters ldquoAirway remodeling contributesto the progressive loss of lung function in asthmaAn overviewrdquoThe Journal of Allergy and Clinical Immunology vol 116 no 3pp 477ndash486 2005

[32] G M Donovan ldquoInter-airway structural heterogeneity inter-acts with dynamic heterogeneity to determine lung functionand flow patterns in both asthmatic and control simulatedlungsrdquo Journal of Theoretical Biology vol 435 pp 98ndash105 2017

[33] S Siddiqui M Novali K Tsuchiya et al ldquoThe modulation oflarge airway smoothmuscle phenotype and effects of epidermalgrowth factor receptor inhibition in the repeatedly allergen-challenged ratrdquo American Journal of Physiology-Lung Cellularand Molecular Physiology vol 304 no 12 pp L853ndashL862 2013


[34] L Auger S Mailhot-Larouche F Tremblay M Poirier CFarah and Y Bosse ldquoThe contractile lability of smooth musclein asthmatic airway hyperresponsivenessrdquo Expert Review ofRespiratory Medicine vol 10 no 1 pp 19ndash27 2016

[35] C Huang Z Zhang L Wang J Liu X Gong and C ZhangldquoML-7 attenuates airway inflammation and remodeling viainhibiting the secretion of Th2 cytokines in mice model ofasthmardquo Molecular Medicine Reports vol 17 no 5 pp 6293ndash6300 2018

[36] C Perkins N Yanase G Smulian et al ldquoSelective stimulation ofIL-4 receptor on smooth muscle induces airway hyperrespon-siveness in micerdquo The Journal of Experimental Medicine vol208 no 4 pp 853ndash867 2011

[37] L Jia PDelmotte BAravamudanCMPabelick Y S Prakashand G C Sieck ldquoEffects of the inflammatory cytokines TNF-120572 and IL-13 on stromal interaction molecule-1 aggregation inhuman airway smooth muscle intracellular Ca2+ regulationrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol49 no 4 pp 601ndash608 2013

[38] Y Chiba G Tanoue R Suto et al ldquoInterleukin-17A directlyacts on bronchial smooth muscle cells and augments thecontractilityrdquo Pharmacological Reports vol 69 no 3 pp 377ndash385 2017

[39] Y Wu H Fu H Yang et al ldquoSmooth muscle progenitorcells involved in the development of airway remodeling in amurinemodel of asthmardquo Asian Pacific Journal of Allergy andImmunology vol 32 pp 203ndash210 2014

[40] L P Desai Y Wu R S Tepper and S J Gunst ldquoMechanicalstimuli and IL-13 interact at integrin adhesion complexes toregulate expression of smooth muscle myosin heavy chain inairway smooth muscle tissuerdquo American Journal of Physiology-LungCellular andMolecular Physiology vol 301 no 3 pp L275ndashL284 2011

[41] O S Matusovsky E M Nakada L Kachmar E D Fixman andA-M Lauzon ldquoCD4+ T cells enhance the unloaded shorteningvelocity of airway smooth muscle by altering the contractileprotein expressionrdquo The Journal of Physiology vol 592 no 14pp 2999ndash3012 2014

[42] P BNoble CD Pascoe B Lan et al ldquoAirway smoothmuscle inasthma linking contraction and mechanotransduction to dis-ease pathogenesis and remodelingrdquo Pulmonary PharmacologyampTherapeutics vol 29 pp 96ndash107 2014

[43] F R Gil and A-M Lauzon ldquoSmooth muscle molecularmechanics in airway hyperresponsiveness and asthmardquo Cana-dian Journal of Physiology and Pharmacology vol 85 no 1 pp133ndash140 2007

[44] G Andre-Gregoire F Dilasser J Chesne et al ldquoTargeting ofRac1 prevents bronchoconstriction and airway hyperrespon-sivenessrdquo The Journal of Allergy and Clinical Immunology vol142 no 3 pp 824ndash833 2018

[45] Y Ren X Su L Kong et al ldquoTherapeutic effects of histonedeacetylase inhibitors in amurine asthmamodelrdquo InflammationResearch vol 65 no 12 pp 995ndash1008 2016

[46] J Xie X Long L Gao et al ldquoRespiratory syncytial virusnonstructural protein 1 blocks glucocorticoid receptor nucleartranslocation by targeting ipo13 and may account for glucocor-ticoid insensitivityrdquo The Journal of Infectious Diseases vol 217no 1 pp 35ndash46 2018

[47] Z Ye L Ren Z Tang et al ldquoPulmonary C-fiber degenerationdownregulates IFN-120574 receptor 1 via IFN-120572 induction to atten-uate RSV-induced airway hyperresponsivenessrdquo Virology vol510 pp 262ndash272 2017

[48] N Zhou W Li L Ren X Xie and E Liu ldquoAn Interaction ofLPS and RSV Infection in Augmenting the AHR and AirwayInflammation in Micerdquo Inflammation vol 40 no 5 pp 1643ndash1653 2017

[49] X Long J Xie K Zhao et al ldquoNK cells contribute to persistentairway inflammation and AHR during the later stage of RSVinfection in micerdquo Medical Microbiology and Immunology vol205 no 5 pp 459ndash470 2016

[50] C Niu T WangW Zou et al ldquoEnhanced pause correlates withairway neutrophils and airway-epithelial injury in asthmaticmice treated with dexamethasonerdquo Journal of Asthma amp AllergyEducators vol 5 pp 1ndash10 2018

[51] S R Singh A Sutcliffe D Kaur et al ldquoCCL2 release by airwaysmooth muscle is increased in asthma and promotes fibrocytemigrationrdquo Allergy vol 69 no 9 pp 1189ndash1197 2014

[52] I Bara A Ozier P-O Girodet et al ldquoRole of YKL-40 inbronchial smooth muscle remodeling in asthmardquo AmericanJournal of Respiratory and Critical Care Medicine vol 185 no7 pp 715ndash722 2012

[53] B G Oliver and J L Black ldquoAirway smooth muscle andasthmardquo Allergology International vol 55 no 3 pp 215ndash2232006

[54] O SMatusovsky LKachmarG Ijpma et al ldquoPeripheral airwaysmooth muscle but not the trachealis is hypercontractile in anequine model of asthmardquo American Journal of Respiratory Celland Molecular Biology vol 54 no 5 pp 718ndash727 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


tissues as revealed by immunohistochemistry and Westernblot analysis The expression of smooth muscle-specific genes(actin-1205722-smooth-muscle-aorta Acta2 and myosin-heavy-chain-11-smooth-muscle Myh11 respectively) was remark-ably increased in neonatal S pneumoniae pneumonia groupconsistent with the Western blotting and histologic resultsFurthermore AHRwas also remarkably higher in the S pneu-moniae pneumonia group compared to the controls Takentogether neonatal S pneumoniae pneumonia promoted anaberrantASMphenotype andAHRdevelopment absent fromallergen challenge in mice model

2 Materials and Methods

21 Establishment of the Non-Lethal Neonatal PneumoniaModel Using S pneumoniae All experimental protocols wereapproved by the Institutional Animal Care and ResearchAdvisory Committee of the Chongqing Medical UniversityThe animals were treated in accordance with the guidelinesissued by the Chinese Council on Animal Care ParturientBALBC mice were purchased from Animal Resources Cen-tre of Chongqing medical university housed separately andclosely monitored for births Newborn mice were housed at25∘Cunder a 12 h lightdark cycle and providedwith adequatefood and waterWe established a non-lethal neonatal S pneu-moniae model of pneumonia in these neonates as describedin our previous study [14] Briefly S pneumoniae D39 strainwas inoculated into tryptic soy broth (Pangtong China) andcultured for 12-14h at 37∘C under 5 CO

2 Neonatal (1-

week-old) BALBC mice were infected intra-nasally with 2times 106 colony-forming units (CFU) of S pneumonia in 5120583lphosphate buffered saline (PBS) while the mock-infectedcontrols received the same volume of sterile PBS Five weekspostinfection the lungswere removed and homogenized andthe tissue homogenates were cultured on blood agar for 24 h(at 37∘C under 5 CO

2) to determine the bacterial load

22 Histology of the Lungs Five weeks after pneumoniaie when the neonates had reached adulthood the micewere euthanized with a lethal dose of 10 chloralhydrate(03ml100g intraperitoneally) and their lungs were har-vested The left lungs were desiccated and embedded inparaffin after fixing in 4 paraformaldehyde Tissue sections(4120583m thick) were stained with hematoxylin and eosin (HampESigma-Aldrich) and the lung lesions were semiquantitativelyscored as described previously [15] 0 points for no cell 1 pointfor few cells 2 points for a ring of inflammatory cells 1 celllayer deep 3 points for a ring of inflammatory cells 2 to 4cells deep 4 points for a ring of inflammatory cells of gt4 cellsdeep

23 Bronchoalveolar Lavage Fluid (BALF) Analysis Follow-ing euthanization of the mice as described above the BALFwas obtained by washing the lungs five times with 1ml ice-cold PBS The number of total cells differential cell countsand the level of cytokines including IL-4 IL-5 IL-13 IL-17A and INF-120574were determined as previously described [14]The cytokine levels were determined using specific enzyme-linked immunosorbent assay (ELISA) kits (Neobioscience

Shenzhen China) according to the manufacturerrsquos instruc-tions

24 Immunohistochemistry (IHC) The lung tissue sectionswere deparaffinized and dehydrated as per standard pro-tocols After blocking endogenous peroxidase activity andnon-specific staining with H

2O2and 5 bovine serum

albumin (BSA) respectively the sections were incubatedwith mouse monoclonal anti-120572-SMA rabbit monoclonalanti-SMMHC or rabbit monoclonal anti-smooth muscle 22alpha (SM22120572) antibodies (all diluted 1100 Sigma-AldrichSt Louis MO) at 4∘C for 12 h The sections were washedwith PBS and incubated with secondary antibody for 30minat 37∘C The positive signals were developed using the 331015840-diaminobenzidine (DAB) chromogen and the sections werecounterstained with hematoxylin The basement membraneperimeter (Pbm) and the 120572-SMA+ SMMHC+ and SM22120572+areas [16] were outlined and analyzed with the Image-ProPlus 60 software (Image-Pro 60 USA) For accurate mor-phometric quantification Pbm2 was first used to standardizethe region of interest ie sections of the airway wall andconnective tissue attachments excluding the blood vessels[16ndash18] The 120572-SMA+ areaPbm2 SMMHC+ areaPbm2 andSM22120572+ areaPbm2 ratios were calculated to respectivelydefine the 120572-SMA+ area SMMHC+ area and SM22120572+ areaaround the airway [17 19]

25 Western Blotting Total protein was extracted from theharvested lungs of adult mice and denatured with theSDS-PAGE loading buffer Equal amounts of protein fromeach group were separated on 10 SDS-PAGE and thentransferred onto polyvinylidene difluoride (PVDF) mem-branes (Millipore) The membranes were incubated withmouse anti-120572-SMA (1500 Sigma) rabbit anti-SMMHC(11000 Sigma) rabbit anti-SM22120572 (11000 Sigma) or rab-bit anti-GAPDH (11000 Proteintech) antibody for 12 h at4∘C After washing with PBS the membranes were incu-bated with the secondary antibody and the positive bandswere quantified using Quantity-One software relative toGAPDH

26 Real-Time Quantitative PCR (RT-qPCR) Total RNAwasextracted from the lungs of adult mice with TRIzol (Invit-rogen CA) and reverse transcribed into cDNA using thePrimeScript RT kit (TaKaRa Japan) The relative expressionsof 120572-SMA gene (Acta2) SMMHC gene (Myh11) and SM22120572gene (transgelin Tagln) were detected using the RT-qPCRassay kit from Life Technologies The sequences of therespective forward (F) and reverse (R) primers are as follows

Acta2-F 51015840-TGCTGGACTCTGGAGATGGTGTG-31015840

Acta2-R 51015840-CGGCAGTAGTCACGAAGGAAT-AGC-31015840

Myh11-F 51015840-CCATTGCCGACACAGCCTACAG-31015840

Myh11-R 51015840-GGATGCCACCACAGCCAAGTAC-31015840

Tagln-F 51015840-AGATGGAACAGGTGGCTCAATTCT-TG-31015840


1 2 3 4 5 6 7 140

1000

2000

3000

4000


pneu

mon

iae c

fum

lSt

rept

ococ

cus

in lu

ng h

omog

enat

e

(a)

1 2 3 4 5 6 7 14 21 28000

005

010

015

020

controlSpp

days post infection

Lung

wei

ght (

g)

lowastlowastlowast

lowastlowast

(b)

1 2 3 4 5 6 7 14 21 280

40

controlSpp

days post infection

Body

wei

ght (

g)

10

20

30



(c)







3 Results






Control Spp

-SMA

(a)

-S

MA

+are

aPb

m

Control Spp0000

0005

0010

0015 lowastlowast

(b)

control Spp0

1

2

3

4

5

lung

tiss

ueA

cta2

mRN

AG

APD

H in

lowastlowast

(c)

GAPDH

Control Spp

-SMA

(d)

Control Spp00

05

10

15

20

-S

MA

pro

tein

GA

PDH

lowastlowast

(e)

SMMHC

Control Spp

(f)

SMM

HC

+ar

eaPbm

Control Spp000

001

002

003

004

005lowast

(g)

Control Spp0

1

2

3

lung

tiss

ueM

yh11

mRN

AG

APD

H in

lowastlowast

(h)

SMMHC

GAPDH

Control Spp

(i)

Control Spp00

05

10

15

20

SMM

HC

prot

ein

GA

PDH

lowastlowast

(j)

Control Spp

SM22

(k)

SM22

+

area

Pbm

Control Spp0000

0005

0010

0015

0020

0025

(l)

Control Spp00

05

10

15

20

lung

tiss

ueTa

gln

mRN

AG

APD

H in

(m)

Control Spp

GAPDH

SM22

(n)

Control Spp00

05

10

15

20

SM22

p

rote

inG

APD

H

(o)


HampE stain

Control Spp

(a)

Control Spp0

1

2

3

peri

-bro

nchi

ole i

nflam

mat

ion

His

tolo

gica

l sco

re o

f

lowastlowastlowast

(b)

Control Spp0

1

2

3

peri

-vas

cula

r infl

amm

atio

nH

istol

ogic

al sc

ore o


(c)

Control Spp0

1

2

3

His

tolo

gica

l sco

re o

f alv

eola

rin

flam

mat

ion

lowastlowast

(d)















4 Discussion


0 3125 625 125 25 500

1

2

3

4

5

ControlSpp

Penh

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast










5 Conclusions


Abbreviations


Data Availability




Acknowledgments


References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















1 2 3 4 5 6 7 140

1000

2000

3000

4000


pneu

mon

iae c

fum

lSt

rept

ococ

cus

in lu

ng h

omog

enat

e

(a)

1 2 3 4 5 6 7 14 21 28000

005

010

015

020

controlSpp

days post infection

Lung

wei

ght (

g)

lowastlowastlowast

lowastlowast

(b)

1 2 3 4 5 6 7 14 21 280

40

controlSpp

days post infection

Body

wei

ght (

g)

10

20

30



(c)







3 Results






Control Spp

-SMA

(a)

-S

MA

+are

aPb

m

Control Spp0000

0005

0010

0015 lowastlowast

(b)

control Spp0

1

2

3

4

5

lung

tiss

ueA

cta2

mRN

AG

APD

H in

lowastlowast

(c)

GAPDH

Control Spp

-SMA

(d)

Control Spp00

05

10

15

20

-S

MA

pro

tein

GA

PDH

lowastlowast

(e)

SMMHC

Control Spp

(f)

SMM

HC

+ar

eaPbm

Control Spp000

001

002

003

004

005lowast

(g)

Control Spp0

1

2

3

lung

tiss

ueM

yh11

mRN

AG

APD

H in

lowastlowast

(h)

SMMHC

GAPDH

Control Spp

(i)

Control Spp00

05

10

15

20

SMM

HC

prot

ein

GA

PDH

lowastlowast

(j)

Control Spp

SM22

(k)

SM22

+

area

Pbm

Control Spp0000

0005

0010

0015

0020

0025

(l)

Control Spp00

05

10

15

20

lung

tiss

ueTa

gln

mRN

AG

APD

H in

(m)

Control Spp

GAPDH

SM22

(n)

Control Spp00

05

10

15

20

SM22

p

rote

inG

APD

H

(o)


HampE stain

Control Spp

(a)

Control Spp0

1

2

3

peri

-bro

nchi

ole i

nflam

mat

ion

His

tolo

gica

l sco

re o

f

lowastlowastlowast

(b)

Control Spp0

1

2

3

peri

-vas

cula

r infl

amm

atio

nH

istol

ogic

al sc

ore o


(c)

Control Spp0

1

2

3

His

tolo

gica

l sco

re o

f alv

eola

rin

flam

mat

ion

lowastlowast

(d)















4 Discussion


0 3125 625 125 25 500

1

2

3

4

5

ControlSpp

Penh

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast










5 Conclusions


Abbreviations


Data Availability




Acknowledgments


References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Control Spp

-SMA

(a)

-S

MA

+are

aPb

m

Control Spp0000

0005

0010

0015 lowastlowast

(b)

control Spp0

1

2

3

4

5

lung

tiss

ueA

cta2

mRN

AG

APD

H in

lowastlowast

(c)

GAPDH

Control Spp

-SMA

(d)

Control Spp00

05

10

15

20

-S

MA

pro

tein

GA

PDH

lowastlowast

(e)

SMMHC

Control Spp

(f)

SMM

HC

+ar

eaPbm

Control Spp000

001

002

003

004

005lowast

(g)

Control Spp0

1

2

3

lung

tiss

ueM

yh11

mRN

AG

APD

H in

lowastlowast

(h)

SMMHC

GAPDH

Control Spp

(i)

Control Spp00

05

10

15

20

SMM

HC

prot

ein

GA

PDH

lowastlowast

(j)

Control Spp

SM22

(k)

SM22

+

area

Pbm

Control Spp0000

0005

0010

0015

0020

0025

(l)

Control Spp00

05

10

15

20

lung

tiss

ueTa

gln

mRN

AG

APD

H in

(m)

Control Spp

GAPDH

SM22

(n)

Control Spp00

05

10

15

20

SM22

p

rote

inG

APD

H

(o)


HampE stain

Control Spp

(a)

Control Spp0

1

2

3

peri

-bro

nchi

ole i

nflam

mat

ion

His

tolo

gica

l sco

re o

f

lowastlowastlowast

(b)

Control Spp0

1

2

3

peri

-vas

cula

r infl

amm

atio

nH

istol

ogic

al sc

ore o


(c)

Control Spp0

1

2

3

His

tolo

gica

l sco

re o

f alv

eola

rin

flam

mat

ion

lowastlowast

(d)















4 Discussion


0 3125 625 125 25 500

1

2

3

4

5

ControlSpp

Penh

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast










5 Conclusions


Abbreviations


Data Availability




Acknowledgments


References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
































4 Discussion


0 3125 625 125 25 500

1

2

3

4

5

ControlSpp

Penh

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast










5 Conclusions


Abbreviations


Data Availability




Acknowledgments


References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























5 Conclusions


Abbreviations


Data Availability




Acknowledgments


References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of













































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Neonatal Streptococcus pneumoniae Pneumonia Induces an ...downloads.hindawi.com/journals/bmri/2019/1948519.pdf · ResearchArticle Neonatal Streptococcus pneumoniae Pneumonia Induces