CCL3 is highly expressed in NEC intestinal lesions and aggravates the intestinal damage
To determine the expression of CCL3 in NEC, intestinal tissue from NEC patients undergoing surgery was homogenized for ELISA test. The results showed that the level of CCl3 in the homogenized necrotic intestinal tissue of NEC patients was significantly higher than that of the surgically excised, relatively non-necrotic adjacent sites, as shown in Fig.1A. Then, an NEC animal model was established for further study, and both ELISA results (Fig.1B) and immunohistochemical results (Fig.1C) showed that CCL3 expression in NEC mice was significantly higher than that in the control group. To characterize the role of CCL3 in NEC, mice in the intervention group were intraperitoneally injected with the recombinant CCL3 protein (rCCL3) or CCL3 neutralizing antibody (anti-CCL3) during modeling and maintained at an appropriate dose after modeling, while mice in the control group were intraperitoneally injected with PBS. Intestinal gross appearance (Fig.1D) and pathological results (Fig.1E) showed that the intestinal tissue damage in rCCL3 group was more severe than that in the control group, accompanied by severe intestinal flatulence and bleeding (Fig.1D), as well as necrotizing changes such as intestinal epithelial shedding and intestinal mucosal thinning (Fig.1E), all of which were significantly alleviated in the anti-CCL3 group. These results indicate that the increased expression of CCL3 is a harmful endogenous factor in the development of NEC.
CCL3 promotes inflammation and macrophage recruitment in the intestinal tissues of NEC mice.
Considering that CCL3 aggravates intestinal tissue damage during NEC, we further evaluated the effects of CCL3 on intestinal tissue inflammation and leukocyte infiltration. ELISA results showed that inflammatory cytokines IFN-γ and IL-1β were significantly increased in the rCCL3 treatment group, but significantly decreased in the anti-CCL3 treatment group (Fig.2A and B). The intestinal tissues of model mice were then digested to prepare single-cell suspensions for the detection of leukocyte infiltration. FCM revealed a significant increase in CD11b+F4/80+ macrophages in the intestine of NEC mice injected with rCCL3, which was reversed by anti-CCL3 treatment (Fig. C and D). CCL3 can also promoted the recruitment of a small number of CD11b+LY/6G+ neutrophils (Fig. E and F), but has little effects on the accounts of lymphocytes (CD3, CD4, CD8) (Fig. G-K). Next, primary peritoneal macrophages (PMφ) were isolated and treated with rCCL3 or anti-CCL3, and culture supernatants were collected for the detection of inflammatory factors. IFN-γ and IL-1β levels in the supernatant of rCCL3 treatment group were significantly increased, while those in anti-CCL3 treatment group decreased (Fig. L and M), which was consistent with the results of in vivo experiments, further confirm the regulation of CCL3 on macrophages in NEC. These results suggest that CCL3 mediates macrophage chemotaxis and participates in NEC-related intestinal inflammation.
Transcriptomics reveal the proinflammatory profile of CCL3-stimulated macrophages
To further clarify the role of CCL3-activated macrophages in NEC, bone marrow derived macrophages (BMDM) were isolated and the spectral characteristics of rCCL3 and anti-CCL3-stimulated macrophages were evaluated by high-throughput sequencing. The volcano map of differentially expressed genes (DEGs) showed significant differences in gene expression between the CCL3-treated group, the anti-CCl3-treated group and control groups (Fig.3 A). To further analyze the DEGs results, Venn diagrams were used to describe the overlap of upregulation genes, and the results showed a higher rate of difference in DEGs between the control and rCCL3 groups than between the control and anti-CCL3 groups (Fig.3 B). Each small square on the heat map represents a DEGs, and all genes are listed in the supplementary material (Supplementary data 1). Importantly, cluster analysis revealed that CCL3 treatment has substantial impacts on the pro-inflammatory response of macrophages, as some inflammatory cytokines (NLRP3, TNF, IL-1B, IL-6, IL-12), chemokines (CCL5, CXCL10, CCL2, CCL7), and M1-type macrophage-related genes (iNOS, CD38, CD86) were highlighted in DEGs of rCCL3-treated macrophages (Fig.3 C), and the representative highlighted DEGs in rCCL3 group were summarized in Table 1. To further specify the functional regulation of CCL3 on macrophages, GO analysis was performed thereafter. DEGs were divided into three categories: biological process (BP), molecular function (MF) or cellular component (CC). Significant differences in GO analysis of DEGs in different groups were defined as P<0.05. Notably, functional annotations by GO analysis indicated that DEGs in rCCL3 pretreatment group were mainly were associated with positive regulation of immune response, positive regulation of cytokine production and proliferation of leukocytes, etc. (Fig.3 F-H). To identify the relevant biological pathways involved in these effects, sequencing data were further analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 3D) and the Reactome Signaling Pathway database (Fig.3 E) according to the criteria of P<0.05. The data suggested that major pathways of DEGs after rCCL3 stimulation included chemokine signaling pathway, and cytokine-cytokine receptor interaction, et al. Based on the analysis of these data, we conclude here that macrophages treated with rCCL3 were highly expressing substantial pro-inflammatory factors associated with the M1 phenotype, whereas the anti-CCL3 group showed the opposite results.
Table3 Partial representative DEGs highly expressed in rCCL3 group
Gene ID
|
Gene name
|
Log2FC
|
P-Value
|
ENSMUSG00000027398
|
Il1b
|
8.625474027
|
<10^-5
|
ENSMUSG00000020826
|
Nos2
|
8.393199099
|
<10^-5
|
ENSMUSG00000029084
|
CD38
|
7.463980027
|
<10^-5
|
ENSMUSG00000035042
|
Ccl5
|
6.738212218
|
<10^-5
|
ENSMUSG00000024401
|
Tnf
|
4.370028163
|
<10^-5
|
ENSMUSG00000025498
|
Irf7
|
3.148264233
|
<10^-5
|
ENSMUSG00000032691
|
Nlrp3
|
3.120521887
|
<10^-5
|
ENSMUSG00000051439
|
Cd14
|
2.477026117
|
<10^-5
|
ENSMUSG00000018459
|
Slc13a3
|
2.311923179
|
2.02E-74
|
ENSMUSG00000022901
|
Cd86
|
1.275628056
|
1.45E-10
|
Table 3 Partial representative data and detailed data can be obtained from the authors for reasonable reasons.
CCL3 mediates M1 macrophage polarization in intestinal tissue of NEC.
Since RNA-Seq data have identified that CCL3 regulate macrophage into a pro-inflammatory phenocyte, we then sought to examine the expression of different phenotypes of macrophage-related genes in intestinal tissues of patients and NEC mice. Compared with the control group, the expression of iNOS and CD86, two representative marker of M1 macrophages, was higher in the necrotic intestinal tissue of NEC patients, while the expression of M2-related genes Arg-1 [20]was lower (Fig.4 A-C). In NEC mouse model, the expression of M1 macrophage-related genes iNOS, CD86 and IRF5 [21]was also increased in rCCL3-treated mice compared with the control group, consistent with that of human results (Fig.4 D-G). On the contrary, the expression of M2 macrophage-related genes Arg1, FIZZ1 and YM1 [22]decreased in rCCL3-treated mice compared with the control group (Fig.4 H-J). Unsurprisingly, the anti-CCL3 group showed the opposite results to the rCCL3 group (Fig.4 E-J). Then, intestinal tissue from mice in different treatment groups was digested to prepare single-cell suspensions for the detection of macrophage phenotype. FCM results showed a significant increase in F4/80+CD86+ cells in the intestine of NEC mice treated with rCCL3, which was not observed in the anti-CCl3 group (Fig.4 K-L). Together, these data suggest that CCL3 promotes polarization of M1-type macrophages in the intestinal tissues of NEC patients and mice.
CCL3 promotes M1 macrophage polarization in PMφ and BMDM in vitro.
We then sought to verify the regulatin of CLL3 on macrophage phenotype by using peritoneal macrophages (PMφ) and BMDMs in vitro. FCM was used to detect the phenotype of PMφ treated with rCCL3 and anti-CCL3. The results showed that the proportion of F4/80+CD86+ cells in rCCL3-treated cells increased, while the proportion of F4/80+CD206+ cells decreased; The opposite was observed in the anti-CCl3 treated cells. In addition, in GM-CSF and rIL-4 stimulated BMDMs, the proportion of F4/80+CD86+ cells was also increased with CLL3 treatment, while no significant difference was observed in the proportion of F4/80+CD206+ cells with/without CCL3 treatment. Intriguely, both M1-related genes and M2-related genes were highly expressed in M2 macrophages after CCL3 treatment, indicating that CCL3 promoted the transition from M2 to M1 macrophages. Furthermore, we assessed the change of M1 macrophage-related genes and M2 macrophage-related genes, and found that PMφ and BMDM in the CCL3-treated group highly expressed M1 macrophage-related genes, but low expressed M2 macrophage-related genes, which was highly consistent with the results of the in vivo experiment. These evidences supported that CCL3 can promote the pro-inflammatory phenotype of macrophages, which may be closely related to the aggravation of NEC related intestinal tissue injury. Therefore, blockade of CCL3 may be a potential novel immunotherapy strategy to reduce NEC-related intestinal tissue damage.