Vitamin A supplement after neonatal Streptococcus pneumoniae pneumonia inhibits the progression of experimental asthma by altering CD4+T cell subsets

Studies demonstrated that pneumonia can decrease vitamin A productions and vitamin A reduction/deficiency may promote asthma development. Our previous study showed that neonatal Streptococcus pneumoniae (S. pneumoniae) infection promoted asthma development. Whether neonatal S. pneumoniae pneumonia induced asthma was associated with vitamin A levels remains unclear. The aim of this study was to investigate the effects of neonatal S. pneumoniae pneumonia on vitamin A expressions, to explore the effects of vitamin A supplement after neonatal S. pneumoniae pneumonia on adulthood asthma development. Non-lethal S. pneumoniae pneumonia was established by intranasal inoculation of neonatal (1-week-old) female BALB/c mice with D39. S. pneumoniae pneumonia mice were supplemented with or without all-trans retinoic acid 24 hours after infection. Vitamin A concentrations in lung, serum and liver were measured post pneumonia until early adulthood. Four weeks after pneumonia, mice were sensitized and challenged with OVA to induce allergic airway disease (AAD). Twenty-four hours after the final challenge, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. We stated that serum vitamin A levels in neonatal S. pneumoniae pneumonia mice were lower than 0.7µmol/L from day 2–7 post infection, while pulmonary vitamin A productions were significantly lower than those in the control mice from day 7–28 post infection. Vitamin A supplement after neonatal S. pneumoniae pneumonia significantly promoted Foxp3+Treg and Th1 productions, decreased Th2 and Th17 cells expressions, alleviated airway hyperresponsiveness (AHR) and inflammatory cells infiltration during AAD. Our data suggest that neonatal S. pneumoniae pneumonia induce serum vitamin A deficiency and long-time lung vitamin A reduction, vitamin A supplement after neonatal S. pneumoniae pneumonia inhibit the progression of asthma by altering CD4+T cell subsets.


Results neonatal S. pneumoniae pneumonia significantly decreased lung vitamin A expressions in mice model.
To investigate the effects of neonatal S. pneumoniae pneumonia (S.pp) on vitamin A expressions, vitamin A levels in lung, serum and liver were measured after pneumonia. We showed that vitamin A productions in liver were similar between neonatal S. pneumoniae pneumonia and mock-infection control groups (Fig. 1A). In contrast, neonatal S. pneumoniae pneumonia inhibited serum vitamin A increasing as compared with the control mice. Serum vitamin A levels in neonatal S. pneumoniae pneumonia mice were lower than 0.7 µmol/L from day 2-7 post infection (Fig. 1B). Furthermore, neonatal S. pneumoniae pneumonia mice had significantly lower lung vitamin A expressions than those in the control mice from day 7-28 post infection (Fig. 1C). These results indicated that neonatal S. pneumoniae pneumonia inhibited serum and lung vitamin A increasing, which inducing serum vitamin A deficiency and long-time lung vitamin A reduction in mice model.

Vitamin A supplement after neonatal S. pneumoniae pneumonia suppressed inflammatory cells infiltrate during AAD.
To investigate the effects of vitamin A levels after neonatal pneumonia on asthma development, mice were supplemented with or without all-trans retinoic acid after neonatal S. pneumoniae pneumonia and induced allergic airway disease (AAD) at early adulthood. Before inducing AAD, we first monitored vitamin A concentrations both in serum and in lung after vitamin A supplementation in neonatal S. pneumonia infection mice. Our results indicated vitamin A supplementation after neonatal S. pneumonia infection didn't result in hypervitaminosis A in this study (Fig. 1B,C). As expected, the total inflammatory cells, neutrophils, eosinophils, macrophages and lymphocyte in the infected allergic (S.pp/OVA) group were significantly increased as compared with the mock-infected allergic (OVA) control mice. In contrast, the number of total inflammatory cells, neutrophils, eosinophils, macrophages and lymphocyte was dramatically reduced in the infected and vitamin A supplement allergic (S.pp+VA/OVA) group when compared to the infected allergic (S.pp/ OVA) control mice ( Fig. 2A-E). These results demonstrated that vitamin A supplement after neonatal S. pneumoniae pneumonia significantly alleviated inflammatory cells infiltration during AAD.
Next, we explored the effects of vitamin A supplement after neonatal S. pneumoniae pneumonia on pulmonary histopathology during AAD. Our results demonstrated that neonatal S. pneumoniae pneumonia remarkably increased the accumulation of inflammatory cells around pulmonary alveoli, bronchioles and pulmonary vascular during AAD as compared with the mock-infected control mice. Furthermore, there were fewer inflammatory cells around pulmonary alveoli, bronchioles and pulmonary vascular in S.pp+VA/OVA group as compared with S.pp/OVA control mice (Fig. 3A). The inflammation scores of pulmonary peribronchitis, perivasculitis and alveolitis in the S.pp/OVA group were significantly higher than those in the OVA group. In contrast, the inflammation scores in S.pp+VA/OVA group were remarkably lower than those in the S.pp/OVA mice (P < 0.05) (Fig. 3B-D). Taken together, these results demonstrated that vitamin A supplement after neonatal S. pneumoniae pneumonia remarkably suppressed inflammatory cells infiltration during AAD. and lung (C) in BALB/C mice. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the infected (S.pp) group, n = 7 mice/group, # P < 0.05, ## P < 0.01 as compared with control group, (n = 5-7 mice/group).
Vitamin A supplement after neonatal S. pneumoniae pneumonia affected cytokines productions during AAD. Twenty-four hours after the final challenge, BALF was obtained to detect the cytokines by ELISA. The productions of IL-4, IL-5, IL-13, IL-17A were significantly higher (P < 0.05), while IFN-γ was remarkably lower in the S.pp/OVA group compared with the OVA group (P < 0.01). In contrast, IL-4, IL-5, IL-13, IL-17A productions were significantly decreased accompanied by IFN-γ production increased dramatically in the S.pp+VA/OVA group when compared to the S.pp/OVA mice. There had no significant difference of TGF-β production among OVA, S.pp/OVA and S.pp+VA/OVA groups (Fig. 5).

Discussion
Asthma is one of the most common chronic diseases in children 19 . Epidemiological studies have demonstrated the association between early-life infections and subsequent asthma development 20,21 . It is now well accepted that asthma is a heterogeneous disease with different clinical subtypes. Viral infections have been implicated in asthma pathogenesis as well as exacerbation [22][23][24][25] . Atypical pathogen infection (such as Mycoplasma pneumoniae) also appears to play an important role in the induction and exacerbation of asthma both in children  Whole-body plethysmography in mock-infected, non-allergic (Control), mock-infected, allergic (OVA), neonatal infected, allergic (S.pp/OVA) and vitamin A supplement after neonatal infected, allergic (S.pp+VA/ OVA) mice was conducted 24 h following challenge with methacholine (n = 6-8 mice/group). **P < 0.01, ***P < 0.001 as compared with the control group, ### P < 0.001 as compared with S.pp/OVA group. (2020) 10:4214 | https://doi.org/10.1038/s41598-020-60665-4 www.nature.com/scientificreports www.nature.com/scientificreports/ and adults 26,27 . Recent studies suggest some bacterial infection have important role in asthma pathogenesis 28,29 . Clinical study stated acute episodes of wheezing in some children are closely associated with bacterial infection 26 and S. pneumoniae infection may increase the risk of asthma exacerbation 30 . Our previous study indicated that neonatal S. pneumoniae infection promoted early adulthood allergic asthma development 8 . As clinical study indicated pneumonia decrease vitamin A levels significantly in children under five years old 13 . In this study, we monitored vitamin A levels after neonatal S. pneumoniae pneumonia and investigated the effect of vitamin A supplement post-infection on the development of allergic asthma. Our findings demonstrated that neonatal S. pneumoniae pneumonia induced serum vitamin A deficiency and long-time lung vitamin A reduction in mice model, vitamin A supplement after neonatal S. pneumoniae pneumonia inhibited airway neutrophils and eosinophils recruitment, alleviated airway inflammation and decreased AHR during AAD. Vitamin A supplement not only promoted Foxp3 + Treg and Th1 cells, but also inhibited Th2 and Th17 cells productions, accompanied by IFN-γ productions increased, type II cytokines and IL-17A expressions decreased during AAD. These results indicated that vitamin A supplement after neonatal S. pneumoniae pneumonia inhibit the progression of experimental asthma by altering CD4 + T cell subsets.
Studies indicated S. pneumoniae or other pathogens infection in infants and young children decreased retinol productions [31][32][33] . Here, we showed neonatal S. pneumoniae pneumonia induced serum vitamin A deficiency and long-time lung vitamin A reduction. In contrast, Katherine et al. 9 stated that S. pneumoniae infection hardly affected serum and lung vitamin A levels in adult vitamin A adequate BALB/C mice, which indicating that S. pneumoniae infection at different periods of life may induce different effects on vitamin A expressions. Possible explanations for long-time lung vitamin A reduction after neonatal S. pneumoniae pneumonia include: (1) insufficient vitamin A storage in neonates; (2) increased vitamin A consumption: fast growth in neonates increases the need of vitamin A, repairing the damaged epithelial may increase vitamin A consumption 13 ; (3) vitamin A decreasing may reduce retinol binding protein (RBP) production, leading to decreased mobilization of vitamin A from liver 34 ; (4) ordinary food supply after neonatal pneumonia is insufficient to restore vitamin A concentrations 35 .
Epidemiological studies demonstrate vitamin A deficiency is common in asthmatic patients 18,[36][37][38] . Growing evidence indicate that vitamin A directing immune cell differentiation and inducing allergic disease. Intestinal studies in vivo and vitro showed that sufficient retinoic acid (a kind of metabolites of vitamin A) can promote intestinal regulatory T cells productions 39,40 . Akiko et al. 41 stated the differentiation of Foxp3 + Treg from naïve CD4 + T cell is decreased in vitamin A deficient mice. Animal studies suggest that sufficient vitamin A can suppress Th2 reaction and promote Foxp3 + Treg and Th1 cells productions [42][43][44] . Consistent with these studies, our data demonstrated that neonatal S. pneumoniae pneumonia reduced Foxp3 + Treg and Th1 productions, increased www.nature.com/scientificreports www.nature.com/scientificreports/ Th2 and Th17 cells expressions during AAD, which aggravated allergic inflammation and AHR. Vitamin A supplement after neonatal S. pneumoniae pneumonia inhibits the progression of experimental asthma by altering CD4 + T cell subsets productions.
Studies have reported negative correlation between vitamin A levels and the risk of asthma development 17,45,46 . Sufficient vitamin A inhibits asthma or allergic disease by downregulating oxidative stress 47 , or via direct effects on the immune system 44,[48][49][50] . One study demonstrated dexamethasone combined with vitamin A therapy promoted allergic asthmatic epithelium repair by down-regulating leucine zipper (GILZ) expression and activating MAPK-ERK signaling 51 . However, some studies suggested infants supplemented vitamin A or multivitamin increased the risk of allergic disease 52,53 . In addition, a study in Norwegian adults showed that daily intake of cod liver oil (rich in vitamin A) for ≥1 month significantly increased the incidence of adult-onset asthma 54 . One possible explanation for these inconsistencies is vitamin A supplement in those without vitamin A reduction/ deficiency may lead to hypervitaminosis A 55 . Hypervitaminosis A has been reported to be associated with airway hyperresponsiveness and increase the risk of asthma in mice model 56

. So it is important to determine vitamin A levels before vitamin A supplementation both in basic and clinical studies.
Our study indicated that neonatal S. pneumoniae pneumonia resulted in serum vitamin A deficiency and long-time lung vitamin A reduction. Vitamin A supplementation after neonatal S. pneumoniae pneumonia promoted Foxp3 + Treg and Th1 productions, reduced Th2 and Th17 cells expressions when exposed to the allergen, which decreasing AHR and inflammatory cells infiltration, and eventually inhibit the progression of experimental asthma in adulthood. Our finding may provide a novel strategy for the prevention of allergic asthma induced by S. pneumoniae pneumonia. While further researches are needed to explore the mechanisms of neonatal S. pneumoniae pneumonia induced serum vitamin A deficiency and long-time vitamin A reduction. More studies are needed to clarify whether our results can be extrapolated to other pathogens and other animals.

Materials and Methods
Animals. Parturient BALB/C mice were purchased from Animal Resources Centre, Chongqing medical university. Pregnant mice were kept separately and monitored for births. Newborn female mice were raised in a pathogen-free environment, and housed at 24 °C under a 12 h light, 12 h dark cycle, and given a normal diet and water. All experiments performed in mice were permitted by the Institutional Animal Care and Research Advisory Committee at the Chongqing Medical University. All experimental animals were used in accordance with the guidelines issued by the Chinese Council on Animal Care.

Induction of Allergic airway disease (AAD).
On the basis of the procedures described in our previous study 8 , four weeks after neonatal S. pneumoniae pneumonia (mice have grown-up into early adulthood), mice were divided into the following four groups: mock-infected non-allergic (control), mock-infected allergic (OVA), infected allergic (S.pp/OVA), infected and vitamin A supplement allergic (S.pp+VA/OVA). In order to induce AAD, mice in the OVA, S.pp /OVA, S.pp+VA/OVA groups were sensitized with intraperitoneal injections of 100 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) diluted in 50% aluminum hydroxide gel (Sigma-Aldrich) for a total volume of 200 μL on days 35 and 42. Mice were exposed to 1% OVA aerosols for 30 min/d, from days 49-52. The controls were sensitized and challenged with same volume of sterile PBS at the same time. Within 24 h after the final challenge AAD was assessed (Fig. 7). Each experiment was repeated three times with a sample size of a total of four to eight mice per group.

Measurement of airway hyperresponsiveness (AHR).
AHR was assessed in vivo by measuring the changes in transpulmonary resistance using a mouse plethysmograph and methods previously described 8,[59][60][61] . Briefly, 24 hours after the final challenge, AHR was measured in conscious, unrestrained mice by whole-body plethysmography (Emca instrument; Allmedicus, France). Each mouse was exposed to aerosolized PBS followed by increasing concentrations of aerosolized methacholine (Sigma-Aldrich, St. Louis, Mo. USA) solution (3.125, 6.25, 12.5, 25, and 50 mg/ml; Sigma) in PBS for 3 min and then rested for 2 min. The average Penh for each concentration was calculated from the continuously recorded pressure and flow data for 5 min. Penh is a dimensionless value and correlates with pulmonary airflow resistance. It represents a function of the ratio of peak expiratory flow to peak inspiratory flow and a function of the timing of expiration.
Bronchoalveolar lavage fluid and cell counting. Mice were anesthetized with 10% chloral hydrate (0.1 mL/100 g, i.p), twenty-four hours after the final challenge. Bronchoalveolar lavage fluid (BALF) was obtained through a cannulated trachea by flushing the lungs twice with 1 ml each of PBS. The two aliquots were then pooled to obtain one sample for each mouse. Erythrocytes were lysed, and the remaining cells were centrifuged at 3000 rpm for 5 min. Total cell numbers in the BALF were determined using a standard hemocytometer. Differential cell counts were classified according to the standard morphology and staining characteristics of at least 250 cells per sample. Supernatants were stored at −80 °C.
Histo-pathology of lungs. Twenty-four hours after the final challenge, to harvest the lungs, mice were euthanized by an intraperitoneal injection of a lethal dose of 10% chloral hydrate (0.3 mL/100 g, i.p). After fixing in formaldehyde for 24 hours, lungs were dissected and paraffin-embedded. The lung tissue was cut into thin www.nature.com/scientificreports www.nature.com/scientificreports/ slices four microns thick and then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). At least five bronchi were selected from each mouse based on size (150-350 mm in diameter) for analysis. In order to reduce evaluator bias, the degree of airway inflammatory cell infiltration was scored in a single-blind fashion. Lung lesions were scored semi-quantitatively using a measurement tool as previously described 62 . Images were captured under a Nikon Eclipse E200 microscope connected to a Nikon Coolpix 995 camera (Nikon, Tokyo, Japan). The severity of inflammation was evaluated by assigning a value of 0 point for normal; 1 point for few cells; 2 points for a ring of inflammatory cells 1 cell layer deep; 3 points for a ring of inflammatory cells 2 to 4 cells deep; 4 points for a ring of inflammatory cells of >4 cells deep.

Statistical analysis.
Results were analyzed using GraphPad Prism (version 5.0; GraphPad, La Jolla, CA, USA) and values are expressed as mean ± standard error. Statistical analysis was performed by either one-way analysis of variance (ANOVA) with Tukey's post-test or two-way ANOVA with Bonferroni's post-test. A value of P < 0.05 was considered significant.