The long noncoding RNA-30162 is regulated by commensal microbiota and modulates CCL24 and ARG1 in macrophages

Alveolar macrophages (AMs) are the largest number of innate immune cells in the distal lung. AMs have critical roles in maintaining immunological homoeostasis and host defense in the lung and are inherently suppressive. Immune homeostasis depends on the integrity of microbiome, which contribute to appropriate maturation and priming of the immune system. The absence of commensal microbiota can lead to changes in AM function; however, little is known about the effect of long non-coding RNA (lncRNA) molecules in this process. Here, by lncRNA microarray analysis using AM samples from antibiotic-treated (Abt) mice, we found that treatment with antibiotics resulted in differential expression of numerous lncRNAs in AMs. Target genes of differentially expressed lncRNAs were associated with several biological pathways, including regulation of immune system processes, angiogenesis, cell differentiation, and chemotaxis, among others. Notably, lncRNA-30162 expression levels were up-regulated in AMs from Abt mice. Moreover, knockdown of lncRNA-30162 expression significantly reduced CCL24 and ARG1 levels in macrophages. These findings indicate that microbiome can regulate the expression of lncRNA-30162 in AMs, identifying a molecular mechanism underlying lncRNA-mediated regulation of macrophage functions.


Abstract
Background Alveolar macrophages (AMs) are the largest number of innate immune cells in the distal lung. AMs have critical roles in maintaining immunological homoeostasis and host defense in the lung and are inherently suppressive. Immune homeostasis depends on the integrity of microbiome, which contribute to appropriate maturation and priming of the immune system. The absence of commensal microbiota can lead to changes in AM function; however, little is known about the effect of long non-coding RNA (lncRNA) molecules in this process.

Results
Here, by lncRNA microarray analysis using AM samples from antibiotic-treated (Abt) mice, we found that treatment with antibiotics resulted in differential expression of numerous lncRNAs in AMs. Target genes of differentially expressed lncRNAs were associated with several biological pathways, including regulation of immune system processes, angiogenesis, cell differentiation, and chemotaxis, among others. Notably, lncRNA-30162 expression levels were up-regulated in AMs from Abt mice. Moreover, knockdown of lncRNA-30162 expression significantly reduced CCL24 and ARG1 levels in macrophages.

Conclusions
These findings indicate that microbiome can regulate the expression of lncRNA-30162 in AMs, identifying a molecular mechanism underlying lncRNA-mediated regulation of macrophage functions.

Background
The respiratory tract is a complex mucosal tissue. There are specific commensal microbiota from the nostrils to the lung alveoli (upper respiratory tract and lower respiratory tract) that are regarded as "gatekeepers" of respiratory tract health and play crucial roles in the development of organs and maintenance of immune homeostasis [1].
In the past, researchers considered it established that the lung was sterile; however, with the development of advanced sequencing technology, in vitro culture technology, and bioinformatics, it has been proven that lung tissue contains specific commensal microbiota, primarily Proteobacteria and Firmicutes, like the upper respiratory tract,but community richness of upper respiratory tract was higher than low respiratory tract [2].
Compared with the upper respiratory tract, the lung has low numbers of bacteria; however, small changes in the number or composition of its microbiota can have dramatic effects on host responses to inflammation [3]. There is a clear interplay between the lungs and the intestines. Intestines microbiome protects the lungs from bacterial and viral infections by regulating immune responses. Varieties of lung disorders are strongly linked to dysbiosis of the gut mycobiota. Chronic gastrointestinal diseases have a higher incidence of lung diseases, such as inflammatory bowel disease and irritable bowel syndrome [4,5].
Alveolar macrophages (AMs), situated on the surface of alveolar cavities, are the richest innate immune cells in the distal lung. These cells are critical for maintenance of immunological homoeostasis, and host defenses in the lung and are inherently suppressive [6][7][8]. The function of AMs is strongly influenced by the commensal microbiota, by inducing bacterial killing by AMs, the microbiome enhances early innate immune system defenses against bacterial infection [9]. Also, the commensal microbiota promote bacterial clearance from the lung by stimulating AMs to produce antibacterial activity [10]. In lung ischemia reperfusion, commensal microbiota prime the inflammatory response by inducing cytokine production by AMs [11]. In 2017, we reported that commensal microbiota maintain AM CCL24 production at low levels, thereby generating anti-metastatic tumor activity [12]. Nevertheless, the molecular mechanisms by which commensal microbiota exert their immune-modulating effects on AMs remain unknown.
Long noncoding RNAs (lncRNAs) refer to a genome without protein-coding function, and which more than 200 nucleotides [13]. These molecules contribute to cellular responses, including gene expression involved in epigenetic and cell function control, promotion of hematopoiesis, immune maturation, modulation of pathogen infection, and initiation of autoimmune diseases [14]. Commensal microbiota can influence lncRNA expression, with levels of lncRNAs in heart, hippocampus, liver, lung, spleen, and thymus of conventional and gnotobiotic mice differing significantly from those of germ-free (GF) mice, suggesting that lncRNAs probably associated with microbiome. Furthermore, various microbiota can induce differential expression of lncRNAs [15]. The intestinal microbiota can regulate the expression of lncRNAs in the gut and other metabolic organs, such as fat, muscle, and liver, [16]. Recent studies have identified some lncRNAs associated with macrophages.
Further, the lncRNA, E33, regulates proinflammatory gene expression in macrophages [17], while the expression of inflammatory cytokines by macrophages is negatively regulated by the lncRNA, Malat2, to suppress inflammation [18]. The lncRNA-CCL2, regulates macrophage expression of IL-1β, IL-6, and TNF-α [19]. Further, in pulmonary fibrosis, the lncRNA, Malat1, is a negative regulator of M2 macrophage polarization, and regulates the profibrotic phenotype of macrophages [20]. Therefore, we hypothesized that lncRNAs may have a vital role in the process of regulating AM functions via regulating expression of target genes in the absence of commensal microbiota.
In this study, by using a mouse model treated with antibiotics, we first studied the regulatory role of lncRNAs on AM functions in the absence of commensal microbiota. The resulting data strongly link the commensal microbiota to AMs and lncRNA function. This enabled us to further investigate the specific molecular mechanism by which commensal microbiota regulate AM functions via lncRNAs. We found that lncRNA-30162 regulates the expression of CCL24 and ARG1 in macrophages. These data validate the importance of commensal microbiota for AM function, demonstrating that they have an essential role, and identify a new molecular mechanism by which lncRNA-30162 may regulate macrophage function.

Mice and antibiotic treatment
Female C57BL/6 mice, 4-to 5-week-old, were acquired from the Shanghai Experimental Center of the Chinese Science Academy (Shanghai, China). As mentioned above [21], mice were treated with neomycin sulfate (1 g/L), ampicillin (1 g/L), vancomycin (0.5 g/L), and metronidazole (1 g/L) in their drinking water for 4-6 weeks, which was replaced twice per week. Mice were anesthetized with isoflurane, their eyeball was removed for blooding, then the mice were killed by severing the spinal cords. Animal experiments were approved by the Animal Care and Use Local Ethics Committee. The procedure was carried out according to the Guide for the Care and Use of Laboratory Animals issued by Anhui Medical University.

Segregation of lung mononuclear cells
As mentioned above [22], by density gradient centrifugation, and using 40% and 70% Percoll solution, mononuclear cells were isolated from the lungs.

Purification of AMs
Segregated lung mononuclear cells were stained, subsequently, AMs (F4/80 + CD11c + ) were sorted using a FACS Aria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The purity of separated cells was more than ninety-five percent.

Microarray analysis
Microarray analysis was conducted as previously described [12]. Microarray experiments were implemented by Shanghai Biotechnology Company (Shanghai, China). Principal component analysis was performed using ClustVis [23], with Pareto scaling applied to rows, and singular value decomposition with imputation was used for principal components calculation. Each sample was from sorted AMs from 15 mice.

GO and KEGG pathway analysis
The target genes of lncRNAs were predicted in trans and cis to reveal the potential function of lncRNAs. To predict cis roles (referring to the effect of lncRNAs on adjacent target genes) coding genes 10 kb downstream and upstream of the lncRNA were searched. PCR reactions were conducted using a total of 45 cycles, including a 25 sec melting step at 95 °C, a 30 sec annealing step at 60 °C, and 50 sec of extension at 72 °C. Information of the gene-specific primers is provided in Table 1. Table 1 The primers for each gene detected by real-time PCR   Table 2. After 48 h, cells were harvested for total RNA extraction. Knockdown efficiency was assessed using RT-qPCR. All siRNA sequences were supplied by Shanghai GenePharma Company (Shanghai, China). Table 2 The siRNA sequences for RNA interference down-regulated lncRNAs (42.74%) in the Abt group, with a total of 634 lncRNAs exhibiting ≥ 2 foldchange (Fig. 1D). Hence, antibiotic treatment led to differential expression of numerous lncRNAs in AMs.
Further, GO and KEGG pathway analyses were applied to determine the possible biological functions of the predicted target genes of lncRNAs differentially expressed in AMs from Abt mice. Functional analysis of lncRNAs target genes was performed to search for related GO terms and pathways in both cis and trans (Fig. 2). Identifying cis target genes was based on the principle that the lncRNAs are close to protein-coding genes they regulated in cis. Target genes of differentially expressed lncRNAs in cis are presented in Fig. 2A. The rational for identifying trans target genes was that the functions of lncRNAs can be recognized by co-expression with protein-coding genes they regulated in trans. Target genes regulated by lncRNAs in trans are presented in Fig. 2B. These results indicate that lncRNA target genes were associated with several biological pathways, including regulation of immune system processes, angiogenesis, cell differentiation, and chemotaxis, among others.

LncRNA-30162 expression was up-regulated in AMs from Abt mice
To confirm the results of the microarray analysis, 29 lncRNAs were randomly selected for RT-qPCR verification, including 17 that were up-regulated and 12 that were down-regulated, based on microarray data. All 29 lncRNA RT-qPCR validation assays generated similar fold-change results to those determined by microarray, indicating that the data acquired using gene chips were highly reliable. Among the analyzed genes, the expression levels of lncRNA-30162 were up-regulated in AMs from Abt mice (Fig. 3A, B).
In addition, the RT-qPCR analysis showed that there was no remarkable difference in lncRNA-30162 expression between AMs and the mouse macrophage cell line, RAW264.7 (Fig. 4A). Next, we determined the subcellular localization of lncRNA-30162 in RAW264.7 cells. FISH assays using a specific probe confirmed that lncRNA-30162 was mainly distributed in the cytoplasm of RAW264.7 macrophages (Fig. 4B).
Knockdown of lncRNA-30162 expression levels in RAW264.7 cells significantly reduced levels of

CCL24 and ARG1
As we reported previously, commensal microbiota maintain low level CCL24 production by AMS, mediating anti-metastatic tumor activity [12]. Based on our findings of differential expression of lncRNAs in AMs from Abt mice, and upregulation of lncRNA-30162 expression in AMs from Abt mice compared with those from untreated counterparts, we next investigated whether lncRNA-30162 is involved in the expression of immune-related genes in AMs. RAW264.7 cells were used as an alternative to AMs, and lncRNA-30162 knockdown was performed by transfecting siRNA into RAW264.7 cells. Knockdown efficiency (Fig. 5A) and the expression of related genes were evaluated by RT-qPCR. The results indicated that Ccl24 and Arg1 were down-regulated following lncRNA-30162 knockdown, while levels of Timp1, Igf1, and Mmp12 did not differ obviously (Fig. 5B). These findings were confirmed by ELISA (Fig. 5C, D). Overall, these results indicate that lncRNA-30162 knockdown can down-regulate the expression of CCL24 and ARG1 in RAW264.7 cells.

Discussion
Despite recent progress in understanding the effects of commensal microbiota on AMs [12], the molecular mechanisms by which these effects are exerted remain poorly understood. In this study, gene analysis of AMs from Abt mice was performed, and 634 lncRNAs were identified as differentially expressed in response to antibiotic treatment, including 363 up-regulated and 271 down-regulated lncRNAs (Fig. 1). Furthermore, according to GO and KEGG pathway analysis, target genes of lncRNAs were associated with regulation of immune system, angiogenesis, chemotaxis, and cell differentiation, among other functions (Fig. 2). One of these lncRNAs, lncRNA-30162, was markedly up-regulated by antibiotic treatment. In RAW264.7 cells, lncRNA-30162 expression levels did not differ significantly from that in AMs, and these findings were confirmed by RT-qPCR (Fig. 4A).
Overall, our results show that the absence of commensal microbiota results in differential expression of lncRNAs in AMs, and several lncRNAs are reportedly involved in the regulation of macrophages [26][27][28][29]. Further investigation revealed that lncRNA-30162 knockdown can significantly reduce the expression levels of CCL24 and ARG1 in RAW264.7 macrophages. Thus, our study demonstrates a novel role for lncRNA-30162 in macrophage regulation.
Commensal microbiota have important roles in promoting clearance of bacteria from the lung, and microbiome induce AMs to produce antibacterial activity via the ERK pathway. [10]. Recently, several lncRNAs have been implicated in regulation of immunity and inflammation through their influence on macrophages, including lncRNA E33, lncRNA Malat2, and lncRNA-CCL2 [17][18][19]. RAW264.7 cells treated with high glucose and free fatty acids led to increased expression of lncRNA uc.48 and induced inflammatory and P2 × 7R-mediated immune responses via ROS formation, cytokine secretion, and activation of the ERK signaling pathway [30]; however, to date, there has been no report regarding the relationship among commensal microbiota, lncRNAs, and AM function. In this study, we sought to investigate lncRNAs regulated by commensal microbiota in AMs, and identify those lncRNAs that may participate in regulating macrophage functions. We identified lncRNA-30162 as a potent regulator of macrophages.
Our data also show that lncRNA-30162 knockdown altered the expression level of CCL24 and ARG1 in macrophages. High expression of CCL24 are strongly related to liver metastatic tumors and primary colorectal [31]. Further, low levels of CCL24, derived from AMs, are essential for natural anti-tumor responses in the lung [12]. ARG1 is induced in alternatively activated macrophages (M2), and is involved in tumor immunity, anti-inflammation, and immunosuppression-related diseases [32].
Hence, our data reveal crosstalk between commensal microbiota signaling and macrophages via lncRNA-30162. This may explain why AMs from Abt mice predominantly exhibit M2 activity, like higher expression of CCL24 and ARG1 [12]. Therefore, it is possible that commensal microbiota maintain AM production of low levels of CCL24 and ARG1 by regulating lncRNA-30162; however, further detailed RNA interference experiments using AMs will be required to determine whether lncRNA-30162 participates in regulation of AM function in the absence of commensal microbiota.
In summary, this study provides novel insights into the effects of commensal microbiota on lncRNA expression in AMs. Moreover, we have identified a signaling axis, comprising commensal microbiota-lncRNA30162-CCL24 and ARG1, with a crucial role in regulating immunity and inflammation. Our findings will assist further exploration of the mechanisms by which commensal microbiota regulate lncRNA expression in AMs.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have declared no conflict of interests.   Prediction and functional analysis of lncRNAs target genes in Abt and control mice. In the Abt group, mice were treated with mixed antibiotics for 5 weeks. GO and KEGG enrichment scatter plots for cis a and trans b of lncRNAs target genes.   Knockdown of lncRNA-30162 expression levels in RAW264.7 macrophages significantly reduced levels of CCL24 and ARG1. a siRNA silencing effects were evaluated by RT-qPCR; *P < 0.05, **P < 0.01, ***P < 0.001, compared with Gapdh. b RT-qPCR analysis of relative mRNA levels in RAW264.7 macrophages treated with siRNA targeting lncRNA-30162. c, d ELISA analysis of CCL24 and ARG1 expression levels in RAW264.7 macrophages; *P < 0.05.

Supplementary Files
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