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Caloric restriction attenuates C57BL/6J mouse lung injury and systemic toxicity induced by real ambient particulate matter exposure

Background: Caloric restriction (CR) is known to improve health and extend life span in human beings. The effects of CR on adverse health outcomes in response to particulate matter (PM) exposure and the underlying mechanisms have yet to be defined. Results: Male C57BL/6J mice were fed with CR diet or ad libitum (AL) and exposed to PM for 4 weeks in a real-ambient PM exposure system located at Shijiazhuang, China, with a daily mean concentration (95.77 μg/m³) of PM 2.5. Compared to AL-fed mice, CR-fed mice attenuated PM-induced pulmonary injury and systemic toxicity characterized by reduction in oxidative stress, DNA damage and inflammation. Analysis of RNA sequencing revealed that several pulmonary pathways involved in production of ROS, cytokine production, and inflammatory cell activation were inactivated, while those mediating antioxidant generation and DNA repair were activated in CR-fed mice upon PM exposure. In addition, transcriptome analysis of murine livers revealed that CR led to induction of xenobiotic metabolism and detoxification pathways, corroborated by increased levels of urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) and decreased cytotoxicity measured in an ex vivo assay. Conclusion: These novel results demonstrate, for the first time, that CR in mice confers resistance against pulmonary injuries and systemic toxicity induced by PM exposure. CR led to activation of xenobiotic metabolism and enhanced detoxification of PM-bound chemicals. These findings provide evidence that dietary intervention may afford therapeutic means to reduce the health risk associated with PM exposure. Transcriptomic analysis further corroborated the of to activate protective molecular pathways. Notably, altered xenobiotic metabolism and detoxification triggered by CR facilitate the metabolic activation and detoxification of PM-bound chemicals such as PAHs. These findings provided novel insight into the mechanism by which CR mediates the protective roles against PM-induced toxicity and suggest that CR might represent a powerful approach for intervention against air pollution-related health injury.

Introduction metabolic activation [18]. CR also reduces the incidence of cardiovascular diseases, cancers, immune deficiencies, neurodegeneration and diabetes in humans [19][20][21]. CR displays health benefits by triggering a series of molecular events, including reduction of oxidative damage, acceleration of autophagy, inhibition of inflammation and decreased DNA damage [18,22]. CR reduces oxidative stress by decreasing the production of reactive oxygen species (ROS) and enhancing the capacity of antioxidants, such as increased activity of antioxidant response elements (ARE) and the levels of glutathione (GSH) [23]. CR has been shown to decrease the levels of circulating pro-inflammatory cytokines, enhance immune functions, and attenuate DNA damage by activation of key components of the DNA-repair machinery, such as recruitment of base excision repair factors and activation of p53 signaling [22,[24][25][26]. Moreover, CR could promote antitumor effects and inhibit metastases through the induction of autophagy and the modulation of the immune microenvironment [27,28]. Although CR may impact the reduction in PM-induced pulmonary injuries, the underlying mechanisms and the key pathways involved in regulation of cellular functions and integrity has yet to be elucidated. Table 1 The mean PM2.5 concentration and cumulative burden during the exposure period. Note: a. Estimated cumulative burden = MV × T × CON × DF. MV: minute ventilation (mL/min); T: total exposure time (min); CON: mean concentration (mg/m3); DF: pulmonary deposition fraction (m3), DF is estimated by MPPD 3.04.
To characterize the chemical composition of PM 2.5 , atmospheric PM 2.5 was collected daily and quantitative analysis was conducted for polycyclic aromatic hydrocarbons (PAHs), nitro derivatives of PAHs (nitro-PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo dioxins (PCDDs), metal elements and anions (Table S4-S8). As a result, the average concentration of benzo[a]pyrene (BaP), PCDF, PCDD, chromium (Cr) and arsenic (As) far exceeded the daily limit value of Ambient Air Quality Standards of China (Table   S9). Thus, the location of this animal study was representative of the heaviest PM exposure areas in China.
CR efficiently protected against mouse pulmonary injury induced by PM exposure As described in the Methods, we successfully established a mouse model with caloric restriction (CR). The daily caloric intake was reduced by 40% and maintained throughout the experiment. Compared with ad libitum (AL)-fed mice, CR led to a reduction in body weight (BW) by 67.31 ~ 78.43% (Fig. 1A-1B), in subcutaneous fat mass by 23.30 ~ 32.20% ( Fig. 1C), and in visceral fat by 43.39 ~ 44.32% (Fig. 1D). Notably, CR conferred mice with enhanced mitochondrial function (Fig. 1F) and absent signs of malnutrition or lean mass loss (Fig. 1E).
To assess the effects of CR on pulmonary injury in response to PM exposure, we conducted histological and bronchoalveolar lavage fluid (BALF) analyses in mice (4 groups, n = 20/group). The histopathological examination revealed that PM exposure induced interstitial infiltration of neutrophils, alveolar septal thickening and alveolar hemorrhage in AL-fed mice, whereas moderate pathologic injury was observed in CR-fed mice ( Fig. 2A).
As indicated by the pulmonary injury score (Fig. 2B (IFN-γ, TNFα, IL-1β, and IL-12p70) in BALF, which is linked to the potency of proinflammation and M1 macrophage polarization increased in AL-fed mice. However, the levels of IL-4, IL-10, and TGF-β1, which indicated the anti-inflammatory capacity and M2 macrophage polarization, were notably higher in CR-fed mice exposed to PM. Taken together, these observations demonstrate that CR leads to reduction in oxidative damage, DNA damage, and pro-inflammation, contributing to the protective effects against PMinduced pulmonary injury.
The effects of CR on regulation of pathways involved in the protective effects towards PM exposure Transcriptome profiling of lung tissues was performed to elucidate the molecular mechanisms underlying the effects of CR on PM-induced pulmonary injury. Initial analysis identified 2,299 and 1,928 differentially expressed genes (DEGs) based on the comparison between CRC and ALC (AF control group) and between CRE and ALE (PM exposed group).
Detailed information of the DEGs is shown in Fig. S3. To address the biological relevance of the modifications in the gene signature profiles, we employed IPA software to identify critical biological functions and molecular pathways. As a result, IPA analysis identified approximately 500 biological functions, diseases and toxicological outcomes related to the identified DEG profiles. The most related diseases and biological functions predicted were categorized targeting organ injury and abnormalities, inflammatory response, cell death and survival, cell-to-cell signaling and interaction, and respiratory disease (Fig. 4A, 4B).
Notably, the biological function changed in CR-fed mice, with or without PM exposure, implicating a reduction in inflammation, accumulation, recruitment and activation of immune cells (leukocyte, monocyte, lymphocyte, neutrophils, etc.), and cytotoxicity (necrosis, apoptosis, cytolysis), concomitant with increased cell viability. These signatures in gene expression correspond to the biochemical and histologic changes that are attributable to CR ( Fig. 1-2).
Next, we analyzed canonical pathways associated with the CR-mediated protective effects.
We identified 542 and 528 pathways significantly altered between CRC and ALC groups, and between the CRE and ALE groups, respectively. Of the pathways identified, we paid special attention to the those related to inflammatory response, oxidative stress, DNA damage and xenobiotic metabolism (Fig. 4C, 4D). Notably, we found that CR led to a decline in the activity of several pathways, including acute phase response signaling, Th1 .58% in AL-fed mice following PM exposure, while no significant change was observed in CR-fed mice (Fig. 5E, 5G). In addition, higher concentration of serum GSH was detected in CR-fed mice, reflecting improved redox status in CR-fed mice (Fig. 5G). To assess the degree of DNA damage, we conducted comet assays in peripheral blood cells and found that the olive tail moments in AL-fed mice were extended by 43%. In contrast, there was no difference between the PM exposed and control groups of CR-fed mice (Fig. 5H).
Xenobiotic metabolism plays a vital role in mediating the toxicity of PM exposure [59].
Dietary intake could interact with xenobiotic response elements (XREs) to regulate the expression and activity of metabolizing enzymes, thus modifying the cellular response to xenobiotic stressors [60,61]. respectively. CR was progressive, initiated at 10% restriction during the first week, 25% during the second week, and to 40% for the remainder experimental period, according to the method described previously [45]. The components of AL diet and CR diet were given in Table S1. The food intake and the body weight were recorded daily (Table S2, Fig. 1A-1B). All animal procedures were conducted in accordance with the guidelines of the Animal Care and Protection Committee of Sun Yat-sen University and Hebei Medical University.

Real-ambient PM Exposure
Mice were kept in isolated ventilated cages (IVC) and exposed to PM in a real-ambient PM

RNA Sequencing
For each group, we randomly selected 3 mice for conducting RNA sequencing of mouse lung or liver tissues, 12 mice in total from 4 groups of mice (AL-fed or CR-fed with or without PM exposure We set fold-change greater than 1.5 times, P value lesser than 0.05 as the standard to define differentially expressed genes (DEGs). These DEGs were analyzed by Ingenuity Pathway Analysis (IPA) software (Qiagen, Germany). The increased and/or decreased activity of pathways and functions of the DEGs were defined via IPA. Significant differences were defined as P value was less than 0.01 and the absolute value of the z score was greater than 2.

Examination Of Urinary OH-PAHs
Concentration       Perturbation of canonical pathways were reversed by CR in mouse livers.