The Effects Of LPS Exposure During Early Life On OVA-induced Airway Allergic Inflammation In BBLB/c Mice

Part one The effects of LPS treatment during sensitization on OVA-induced airway allergic inflammation Background: Asthma is a highly heterogeneous chronic inflammatory disease with many endotypes.Among these,allergic asthma is the most common type and also known as asthma.Allergic asthma is mediated mostly by Th2 cells,characterized by airway eosinophilia,elevated levels of serum Ig E,goblet cell hyperplasia and BHR.Hygiene hypothesis proposes that increased risk of asthma in developed countries is due to the reduction of microbial exposure in newborn/infant.The newborn/infant period is regarded as an important “time window” for the development of allergic diseases because of the Th2 tendency.It is believed that the exposures to the pathogens or its relevant ingredients(such as lipopolysaccharide,LPS)during this period affect the sensitization to the allergens.However,the effect of LPS treatment on allergic asthma is still controversial,and most studies were conducted in the adult mice.It remains to be clarified whether LPS plays a protective role in the neonatal allergic asthma model and whether the underlying mechanisms vary with age.Objective: To study the effects of LPS treatment during sensitization in neonatal and adult mice on OVA-induced allergic airway inflammation.i.Mouse allergic asthma model: Sensitization phase: Neonatal(7 days old)and adult(7 weeks old)BALB/c mice were intraperitoneally(i.p.)sensitized with OVA emulsified in aluminum hydroxide on days 0 and 7(the dose was calculated according to body weight;20 μg OVA and 4 mg aluminum hydroxide in 100 μl saline for 20 g body weight).Challenge phase: From days 14 to 18,these mice were challenged with OVA aerosols for 30 min.Mice received saline were served as negative control.ii.LPS treatment: Mice were treated i.p.with 1 μg LPS in 50 μl of saline 1 hour before each OVA/alum sensitization.Mice received saline were served as negative control.iii.Measurement of airway inflammation: Mice were sacrificed 24 hours after the final OVA challenge.Wright-Giemsa staining was used for differential counting of cells recoverd from BALF;ELISA was used to measure the levels of cytokines in the BALF and the levels of Ig E and Ig G1 in the serum;PAS and H&E staining of lung sections was used to detect goblet cell metaplasia and the infiltration of cells;The lung inflammation was scored.iv.Immunophenotyping of lung cells and PBLN cultures: 24 hours after the last challenge,the lung single-cell suspension was prepared and the immune cells were evaluated by FCM.PBLNs were dissected and the cells were restimulated with OVA for 3 days.The concentration of cytokines in the culture supernatants was measured by ELISA.v.Measurement of immune cells and cytokines in the peritoneal cavity: Neonatal and adult mice were injected i.p.with 1 and 5 ml PBS,respectively.The peritoneal lavage fluids(PLF)were collected and centrifuged.FCM was used to measure the composition and phenotype of immune cells in the PLF.ELSIA was used to measure the levels of danger signal(Uric Acid、IL-33 and IL-1β),chemokines(CCL-2、CCL-5、CCL-11 and IL-5)and cytokines(TNF-α、IL-6、IL-12 and IFN-γ)in the PLF.Methods:Results: i.Following OVA sensitization and challenge,both age groups developed typical allergic airway inflammation,characterized by airway eosinophilia,elevated levels of IL-4、IL-5 and IL-13 in the BALF,peribronchial and perivascular inflammatory cells infiltration,goblet cells metaplasia and elevated levels of serum Ig E and Ig G1.When comparing the two age groups,we found that the number of eosinophils,the levels of Th2 cytokines,and OVA-specific Ig E in the neonatal group were higher than those of adult group.ii.Th2-mediated responses in both age groups were significantly attenuated upon LPS treatment.However,the elevated IL-17 A was only seen in the adult group and the decreased peribronchial and perivascular inflammatory cell infiltration was only seen in the neonatal group.iii.LPS treatment significantly reduced the levels of Th2 cytokines and increased the level of IFN-γ in the culture supernatants from PBLN cells restimulated with OVA for 72 hours,while the increased IL-17 A level was only detected in the adult group.iv.At steady state,the composition of immune cells in the peritoneal cavity varies with age.The dominant population was macrophage(67.3%)and B cells(60.2%)in the neonatal group and adult group,respectively.v.LPS treatment has age-dependent effects on inflammatory monocytes in the peritoneal cavity.The significantly reduced number of inflammatory monocytes upon LPS treatment was only seen in the neonatal group.In both age groups,the expression of IFN-γ gene in the monocyte and the levels of IL-6,IL-12 and IFN-γ in the PLF was significantly increased upon LPS treatment.In addition,the number of eosinophils was decreased and the number of neutrophils was increased upon LPS treatment.Conclusions: i.Following OVA sensitization and challenge,both age groups developed canonical airway allergic inflammation,which was more severe in the neonatal group.ii.LPS treatment almost entirely inhibited the development of Th2 airway allergic inflammation in the neonatal groups.In the adult group,LPS treatment attenuated airway allergic inflammation and induced Th17 polarization and neutrophil infiltration.iii.LPS treatment inhibited the Th2-mediated allergic inflammation by reducing the number of inflammatory monocytes in the neonatal mice or by shifting the function of these cells in the adult mice.iv.In the adult group,LPS treatment enhanced the expression of cytokines associated with Th1 and Th17 polarization in the peritoneal cavity.Part two The effects of airway LPS exposure during early life on OVA-induced mice airway allergic inflammation Background: The Previously we have found that LPS exposure in early life inhibited the development of OVA-induced airway allergic inflammation.In the searching of the key molecules that mediate the protective effects,the lung tissue after LPS exposure was collected for RNA sequencing and the expression of immune responsive gene 1(Irg1,encodes IRG1)was found to be significantly increased.IRG1 is responsible for itaconic acid synthesis,and the IRG1/itaconate axis is recently discovered to be an important negative regulator in the "endotoxin tolerance" of macrophages.Therefore,we hypothesized that itaconic acid may be a key molecule in the protective effect of LPS exposure.However,whether itaconic acid can penetrate cell membranes is still controversial.For this reason,we used itaconic acid analogs,Dimethyl itaconate(DMI)during the OVA challenge phase to investigate the effects and underlying mechanisms.Objective: i.To analyze the changes of lung tissue transcriptome upon early life LPS exposure.ii.IRG1 emerged from the transcriptome data.We then investigated the effect of DMI treatment during OVA challenge in the neonatal model and the underlying mechanisms.Methods: i.The LPS exposure model: Neonatal BALB/c mice were exposed to 100 ng LPS(i.n.)every other day for 6 times.9 weeks later,the lung tissues were isolated for RNA sequencing.ii.The allergic asthma model of neonatal BALB/c mice was established asdescribed in part one.iii.DMI treatment: Mice were treated i.p.with DMI 24 hours before the initial OVA challenge and 1 hour before each OVA challenge(the dose was calculated according to body weight).iv.In vivo experiments and measurement: a)Airway inflammation detection: same as described in part I.b)FCM was used to measure the composition and functional of immune cells in the BALF and lung.c)The levels of epithelial-derived cytokines and lactate were measured by ELISA.d)The immunochemistry staining of NRF2 of lung sections was used to evaluated the nuclear accumulation of NRF2 in the lung epithelium.e)Western blot and real time PCR were used to measure the expression of NRF2,HO-1 and Nqo-1 in the lung.v.In vitro experiments and measurement: a)The effect of DMI on M2 polarization of macrophages: Peritoneal macrophages from 3 weeks old BALB/c were isolated,pretreated with 0.25 m M DMI and 1 h later stimulated with rm IL-4(20 ng/ml)for 24 hours.the expression of CD206 was detected by FCM.The expression of M2 polarization markers and transcription factor PPAR-γ was measured by Real time PCR.b)The effect of DMI on T cell activation: Spleen naive CD4+ T cells from 3 weeks old BALB/c were stimulated with anti-CD3/anti-CD28 in the presence of different concentrations of DMI for 24 hours or 72 hours.The expression of CD44 and CD25 was measured by FCM 72 hours after stimulation.The activity of GAPDH in the T cell lysates and the levels of lactate in the culture supernatants was measured 24 hours after stimulation.c)The effects of DMI treatment on Th2 differentiation: Splenic naive CD4+ T cells from 3 weeks old BALB/c were isolated.T cells were differentiated under Th2 polarization condition with different concentrations of DMI for 6 days.The intracellular cytokines were measured by FCM.Results: i.The increased expression of IRG1 in the lung tissue was induced by early life LPS exposure.ii.DMI treatment during OVA challenge significantly inhibited the development of allergic airway inflammation in the neonatal BALB/c mice.iii.DMI treatment significantly inhibited the type 2 responses,demonstrated as the decreased numbers of M2 macrophages,ILC2 and Th2 cells in the lung.The number of PBLN cells was much lower and the levels of Th2 cytokines(IL-4,IL-5,and IL-13)in the culture supernatant were decreased.iv.The number of CD11b+ DCs and the expressions of OX40 L on CD11b+ DCs were much lower in DMI-treated group.v.DMI treatment resulted in increased nuclear accumulation of NRF2 in the lung epithelium,along with enhanced expressions of NRF2,HO-1 and Nqo-1 in the lung.vi.In vitro,DMI treatment reduced the expression of CD206+ and M2 polarization markers(Arg1,Fizz1,Ym1,Mgl1 and Mgl2),accompanied by a decrease in PPAR-γ m RNA expression.vii.In vitro,DMI treatment decreased T cells activation and Th2 differentiation in a dose dependent manner,accompanied by a significant decrease in GAPDH enzyme activity and lactate production.Conclusions: i.Early life LPS exposure activates IRG1/itaconate axis.ii.DMI treatment during OVA challenge attenuated the development of airway allergic inflammation in the neonatal model.iii.In vivo,the effects of DMI were on multi-targets(epithelial cells/ DC/ M2/ Th2),either direct or indirect.iv.In vitro,DMI was shown to have a direct effect on M2 polarization,T cell activation and Th2 differentiation.v.DMI may function through activating NRF2,inhibiting glycolytic pathways,and inhibiting PPAR-γ.