Effect of Chronic Adrenocorticotropine Hormone Treatment on LPS-induced Slight Inflammation via Inhibiting Nuclear Translocation of Glucocorticoid-glucocorticoid Receptor Complex in Lung of Pig

1Department of Animal Husbandry and Veterinary Medicine, Jiangsu Polytechnic College of Agriculture and Forestry, Jurong 212400, P. R. China 2Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, P. R. China 3Key Laboratory of Animal Physiology & Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, P. R. China


INTRODUCTION
T he management and transport of modern farms always induces some persistent stresses in domestic animals. The lipopolysaccharide (LPS) challenge reduced feed consumption and increased plasma pro-inflammatory cytokines of pigs, which was inhibited by high temperature O n l i n e

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the body to produce inflammatory response (Mukesh et al., 2010;Wang et al., 2017). However, little information is available regarding the effect of chronic stress on LPSinduced inflammatory response in the lungs of pigs. It is well known that the hypothalamic-pituitaryadrenal (HPA) axis is involved in the modulation of inflammatory response, and the immune-HPA axis interaction is critical for the maintenance of homeostasis. TLRs are responsible for the recognition of a variety of pathogens and induces inflammatory responses, which are normally restricted by the HPA axis with a feedback mechanism (Chinenov and Rogatsky, 2007). The HPA axis is known to be activated by a variety of bacterial, viral and inflammatory insults, and then releases glucocorticoid (GC), which acts through a ligand-dependent transcription factor-glucocorticoid receptor (GR) (Chinenov and Rogatsky, 2007;Sun et al., 2013). Chronic stress can stimulate the HPA axis to release adrenocorticotropine hormone (ACTH), and the circulating ACTH then stimulates the adrenal gland that releases GC to regulate inflammation (Verbrugghe et al., 2012). It remains uncertain whether chronic stress can interfere with LPSinduced inflammation via GR.
Continuous ACTH treatment for animals could simulate chronic stress (Gamallo et al., 1988) (Haussmann et al., 2000). The present work aims to investigate the effect of chronic stress on LPS-induced inflammatory response in local lung tissue of pig. Therefore, we used a pig model exposed to LPS injection with or without continuous ACTH pretreatment to describe secretions of inflammatory cytokines, enzymes regulating oxidative stress, cortisol, levels of pulmonary TLR2-4, GR mRNA, and protein expression, including expression levels of GR protein in the nucleus and all miRNAs that target TLR2 and TLR4.

Animals and experimental design
A 2×2 factorial design was used for injection with LPS or saline as one factor and ACTH treatment or saline as the other factor. Twenty-four 30-day-old Duroc × Landrace × Large White crossbred piglets with average weights of 12 ± 0.5 kg (obtained from our partner farm, Zhenjiang Muyuan Animal Science and Technology Development Co., Ltd., Zhenjiang, China).) were randomly assigned to 4 treatments: control group (CC), LPS (E. Coli serotype, Sigma Aldrich, Dublin, Ireland) injection without ACTH (Sigma Aldrich, Dublin, Ireland) pretreatment group (LPS), ACTH alone treatment group (ACTH), LPS injection with ACTH pretreatment group (ACTH+LPS). Each group consisted of 6 male piglets.
All pigs were provided with water and food ad libitum. The ACTH pigs and ACTH+LPS pigs were injected intramuscularly with ACTH (2.25 IU/kg body weight) for 7 consecutive injections at 6-h intervals. Pigs in the other two groups were mock-injected with saline in the same manner. The LPS pigs and ACTH+LPS pigs were injected intramuscularly with LPS (15 μg/kg weight) immediately after the seventh ACTH treatment. Pigs in the control and ACTH groups were mock-injected with saline. Then, all pigs were euthanized 6 h post-LPS treatment.

Sample collection
The lobus apicalis of the right lung was removed, and the samples were snap frozen in liquid nitrogen and stored at -70°C. Blood was collected from the precaval vein. Blood samples were centrifuged at 1,500×g for 15 min at 4°C, and the serum samples were stored at -20°C.

Cytokine level in serum
Concentrations of TNF-α and IL-10 in serum were measured in duplicate using a commercial TNF-α enzymelinked immunosorbent assay (ELISA) kit (PTA00, R&D Systems Inc., Minneapolis, MN) and IL-10 ELISA kit (P1000, R&D Systems Inc., Minneapolis, MN) according to the manufacturer's instructions. The detection limits of TNF-α and IL-10 were 2.8 to 5.0 pg/mL and 1.8 to 5.5 pg/ mL, respectively.

Cortisol and IL-6 level in lung tissues
Concentrations of cortisol and IL-6 in the supernatant of lung tissues were measured in duplicate using a commercial 125 I-RIA kit (D10PZB and C12PDB, respectively, Beifang Research Institute of Biotechnology,

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Beijing, China) according to the manufacturer's instructions. The detection limits of cortisol and IL-6 were 2 ng/mL and 50 pg/mL, respectively. All samples were measured in the same assay to avoid inter-assay variations.
The data was collected in Nanjing General Hospital of Nanjing Military Command.

Gene expression level
Briefly, total RNA was extracted from lung tissue using Trizol reagent (15596018, Invitrogen, Carlsbad, CA) according to our previous description (Sun et al., 2013). From each sample 1 μg of total RNA was converted to cDNA using PrimeScript® RT reagent kit with gDNA Eraser (DRR047A, Takara, Dalian, China). The primers used are listed in Table I. Also, QPCR reactions and gene expression levels of mRNA were performed using the SYBR Green QPCR Master Mix (DRR041A, TOYOBO Ltd., Japan) as previously described (Sun et al., 2013). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as a reference gene for normalization.

Protein expression level analysis
As described in our previous work (Sun et al., 2013), nuclear, cytosolic, and total protein were extracted from lung tissues, and the protein expression level was analyzed by using Western blot in the same way. In brief, protein was transferred onto nitrocellulose membranes and incubated overnight at 4°C with a primary antibody GR (sc-1004, Santa Cruz Biotechnology, Santa Cruz, CA) or TLR2 (sc-166900, Santa Cruz Biotechnology, Santa Cruz, CA). Lamin A/C (BS1446, Bioworld, China) or β-actin (KC-SA08, Kangcheng, China) was used as a loading control. The membranes were detected using chemiluminescence.

Statistical analysis
Descriptive statistics were performed to check the normality and homogeneity of variances before using parametric analyses. All data were analyzed by two-way analysis of variance (ANOVA) in the general linear model (GLM) procedure of SPSS 16.0 (SPSS Inc., Chicago, IL, USA) with LPS and ACTH treatments as main effects. For interaction between factors, a one-way ANOVA analysis was performed. Duncan's test was used as the post hoc test. Two-tailed P-values with P ≤0.05 were considered significant. Data are expressed as mean ± SEM.

Serum cytokine level
Treatment with LPS increased levels of TNF-α in serum (P <0.01), but ACTH injection has no significant effect on them. No significant interaction was observed between the two treatments (P >0.05) (Fig. 1). Secretion of IL-10 protein was not detected in serum.

Inflammation and cortisol level in lung
As shown in Table III, LPS treatment activated expressions of IL-1β and IL-10 gene mRNA (P <0.01 and P =0.006, respectively), and ACTH treatment had no significant effect on them. There was no significant interaction between ACTH and LPS treatments for expressions of the two genes. As for other genes related to inflammation, no significant differences were observed in the expression of IL-6, TNF-α, IL-10, and COX-2 mRNAs post ACTH or LPS treatment (P >0.05).
Pulmonary cortisol level was inhibited by LPS (P =0.034), with a similar trend of inhibition by ACTH (P =0.062). However, there was no significant interaction between ACTH and LPS treatments (P >0.05).

Oxidative stress in lung
As shown in Table IV, LPS treatment increased levels of T-NOS, iNOS and cNOS protein secretion (P =0.002, P =0.028 and P =0.003, respectively), but ACTH treatment had no significant effect on them. There was a significant interaction between ACTH and LPS treatments for T-NOS and cNOS (P =0.038 and P =0.01, respectively). Levels of pulmonary SOD, CAT and MDA secretion were not affected by ACTH or LPS treatment (P >0.05).  Levels of pulmonary TLR2, 4 mRNA expression Treatment with LPS increased the level of pulmonary TLR2 mRNA expression (P =0.04), but ACTH had no significant effect on them. There was no significant interaction between the two treatments (P >0.05) (Fig. 2a). Neither ACTH nor LPS had any significant effect on TLR4 mRNA expression (Fig. 2b). Secretion of IL-10 protein was not detected in serum.

Levels of pulmonary GR mRNA and protein expression
As shown in Figure 3b, ACTH pretreatment tended to downregulate the expression of total GR protein (P =0.084), but LPS treatment had no significant effect on it. A downward tendency was observed in nuclear GR protein expression post-LPS pretreatment (P =0.088), but no significant effect was found post ACTH treatment (Fig.  3d). Neither ACTH nor LPS treatment had a significant effect on pulmonary GR mRNA or cytoplasmic GR protein expression ( Fig. 3a and c).

Expressions of miRNA
All miRNAs that target TLR2 and TLR4 were predicted. The result showed that miR-338, miR-146a, miR-146b, and Let-7i had potential regulatory effects on TLR4. As shown in Table V, pulmonary expression level of Let-7i was significantly increased by treatment with ACTH (P = 0.013), but not with LPS. However, neither ACTH nor LPS treatment had any significant effect on the expression of miR-338, miR-146a and miR-146b. No potential miRNA was found for TLR2.

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Z. Sun et al.

DISCUSSION
LPS is known to promote the release of cytokines and stimulate inflammatory response. For example, it has been reported that levels of pulmonary pro-inflammatory cytokines, such as TNF-α, IL-1β, and granulocyte-macrophage colony-stimulating factor, were elevated in mice treated with aerosol LPS (Matteri et al., 1998). In a previous study, concentrations of TNF-α, IL-1β, and IL-6 in mammary tissues were increased after an intramammary infusion of LPS (Miao et al., 2007). Correspondingly, in our pro-inflammatory model, the level of TNF-α in serum was increased after intramuscular injection of LPS, which O n l i n e

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Crosstalk bqetween GC and Immune Response during Inflammation 7 indicated that the whole-body system of pigs was sensitive to LPS treatment. However, in all the cytokines detected in our experiment, LPS only increased concentrations of IL-1β and IL-10 mRNA in lung tissues, while the expression levels of other cytokines remained unchanged. This suggests that there was only a slight inflammatory response in the lungs at this time, which is not consistent with previous reports. This may be due to the different treatment methods of LPS. In previous studies, the lungs or breast tissues were directly treated with LPS; therefore, a strong inflammatory response was caused. However, in our study, the method of LPS treatment was intramuscular injection, which only caused a mild inflammatory response in local lung tissues. It was also reported that the cytokine response induced by LPS was age-and tissue-dependent in neuroendocrine tissues of newborn pigs (Matteri et al., 1998). Therefore, the specificity of age and tissue may have impacted the inflammatory response of pig lungs in our experiment. The inflammatory response was stimulated by LPS mainly through activation of TLR4. Then, TLR4 activates signal transduction pathways, such as nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK), through a series of signal cascade reactions, and ultimately activates the inflammatory response (Barton and Medzhitov, 2003;Nasu and Narahara, 2010). A large number of studies have used LPS to induce pro-inflammatory reactions when studying the mechanisms relative to TLR4 (Juarranz et al., 2006;Kim et al., 2012;Wang et al., 2009;Willis et al., 2010). However, in our experiment, LPS treatment had no significant effect on the expression of TLR4 mRNA but increased the expression of TLR2 mRNA. In fact, LPS can activate the TLR2 pathway. It has been found that knockout of TLR2 can inhibit the pro-inflammatory response of LPS, and decrease the secretion of cytokines, such as IL-1, TNF-α, IL-6, and other cytokines in the adrenal gland and serum (Bornstein et al., 2004). It was also found that TLR2 mRNA expression was upregulated in LPS-induced hemorrhagic shock of mice (Fan et al., 2007). It was reported that TLR4 was approximately 100fold more sensitive to LPS than TLR2 in human embryonic kidney 293 cells, which depended on CD14 and LPSbinding protein (Muta and Takeshige, 2001). A higher level of TLR4 expression was associated with higher sensitivity to LPS, which has also been demonstrated by overexpression of TLR4 in transgenic mice (Levy et al., 2009;Bihl et al., 2003). Therefore, in our study, only TLR2 mRNA was upregulated, which was consistent with a weaker inflammatory reaction. This suggests that the inflammatory reaction in the lung may only be activated by the TLR2 pathway.
Many studies have demonstrated that TLRs were activated in organ injury, which was associated with oxidative stress (Pushpakumar et al., 2017;Lee and Kang, 2017). Various inflammatory cytokines can induce the release of reactive oxygen species and formation of toxic metabolites, such as MDA (Zhu et al., 2017). NO, as an important inflammatory mediator, is produced by iNOS (Li et al., 2017). Here, we found that LPS treatment increased levels of pulmonary T-NOS, iNOS, and cNOS, but had no significant effect on levels of SOD, CAT, and MDA. These results suggest that LPS may only promote the production of NO in local lung tissue but does not induce lipid peroxidation. Meanwhile, ACTH treatment has no significant effect on levels of TLR2, TLR4, cytokine, iNOS, and MDA. All these results suggest that the chronic stress simulated by continuous ACTH intramuscular injection has no significant effect on oxidative stress and inflammatory response in the local lung tissue of pigs. However, we also found that ACTH pretreatment down-regulated LPS-induced expressions of TNF-α, COX-2, TLR2 and TLR4 mRNA, as well as IL-6 protein level in porcine adrenal gland (data not shown). Therefore, we speculated that effect of ACTH on LPSinduced inflammation is tissue-specific.
In the cytoplasm, unliganded GR in the inactivation state is combined with heat shock protein 90 (Pratt et al., 2004). GC is a small hydrophobic molecule, which can easily pass through the cell membrane and combine with GR in the cytoplasm, forming GC-GR complex in the nucleus (Pratt et al., 2004;Bledsoe et al., 2004). The GC-GR complex finally transcribes and regulates the expression of innate immune molecules in the nucleus and plays an anti-inflammatory and anti-immune role. We found that pulmonary cortisol levels and nuclear GR protein expression were inhibited by LPS. This result is consistent with significant upregulation of lung TLR2 mRNA and mild inflammatory response in the lungs. This suggests that LPS may increase the expression of TLR2 by inhibiting the anti-inflammatory effects of GC-GR, thus causing a slight inflammatory response in the lungs of pigs. Here, we also found that ACTH pretreatment had a tendency to downregulate the expression of pulmonary cortisol and total GR protein but had no effect on nuclear GR protein expression. This suggests that GC-GR translocation into the nucleus may not be affected by chronic ACTH treatment, so that inflammatory response in the lungs is not changed.
Many reports on the effects of ACTH treatment on cortisol were not consistent. For example, high doses of ACTH in infantile spasms caused a rapid increase of serum cortisol (Snead et al., 1989). Serum cortisol levels were slightly increased in healthy adults from the third to the fifth day during chronic ACTH treatment,

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but decreased in patients with multiple sclerosis (Maida and Summer, 1979). Plasma steroid levels of guinea pigs receiving chronic ACTH treatment were activated with a brief upregulation after the last injection of ACTH and then returned to the basic level (Provencher et al., 1992). Thus, the effect of ACTH on cortisol release is associated with the species and type of stress. Here, we found that ACTH pretreatment had a tendency to downregulate the expression of pulmonary cortisol levels. Cortisol in the body may be metabolized locally in the tissue by 11β-hydroxysteroid-dehydrogenases 1 and 11β-hydroxysteroid-dehydrogenases 2, which activates and inactivates GCs, respectively (Holmes et al., 2003). Therefore, we speculate that the activity of both enzymes in the lungs was altered by chronic ACTH treatment, resulting in a decrease of pulmonary cortisol levels. Moreover, as we discussed in a previous report, GC/GR interaction is highly dynamic (Sun et al., 2013), and release of GC is rhythmic and pulsating with a peak approximately each hour (Biddie et al., 2012). Therefore, single time point sampling, as used in the present study, may produce highly individual variations due to unsynchronized GC pulsatility. The miRNAs, as a class of endogenous non-coding RNA, can regulate inflammation through inhibiting mRNA transcription or promoting mRNA degradation of target gene. In present study, LPS treatment had no significant effect on expression of all the miRNA predicted. Thus, we speculate that pulmonary inflammation was not mediated by miRNAs post LPS treatment. Previous studies have shown Let-7i, as a member of the let-7 miRNA family, can decrease TLR4 expression and inhibit inflammation and oxidative stress (Qi et al., 2016;Xiang et al., 2017). Here, ACTH significantly increased pulmonary expression level of Let-7i. But inconsistent with expectation, TLR4 mRNA levels were not affected. Actually, a single miRNA can regulate thousands of target genes to control innate and adaptive immune response (Singh et al., 2013). This suggests that ACTH-induced increase of Let-7i expression level may target other gene to modulate immune activity in lung of pig.
GR can take part in modulating mRNA expressions of a series of inflammatory gene via complex mechanisms of transcriptional regulation. It can directly or indirectly regulate target genes by itself or interfering with other transcriptional factors (Beck et al., 2009;Biddie et al., 2012). Our previous experiments have proved that vaccination of inactivated vaccine resulted in diminished TLR2 expression via the inhibition of GR nuclear translocation and binding to the TLR2 promoter (Sun et al., 2013). It has also been showed that GR interfered with the transcriptional activity of NF-κB and Activator protein 1 (AP-1), thus inhibiting the expression of inflammatory molecules such as IL-2, IL-6, IL-8, TNF-α and iNOS (De Bosscher et al., 2003). Therefore, further in vitro investigations are needed to delineate the complex regulatory network of GR and inflammatory molecules post nuclear translocation of GR.

CONCLUSION
Taken together, we first in vivo determined that LPS may increase the expression of pulmonary TLR2 mRNA by inhibiting nuclear translocation of GC-GR, thus causing a slight inflammatory response in the lungs of pigs. ACTH pretreatment-stimulated stress tended to down-regulate pulmonary cortisol and total GR protein levels, but had no effect on LPS-induced slight inflammation in local pig lungs. Our study will deepen the knowledge about the relationship between immune response and stress during inflammation, and may provide a theoretical basis for using this relationship as a useful alert for porcine health and welfare.