IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice

Abstract In schistosomiasis japonica and mansoni, parasite eggs trapped in host liver elicit severe liver granulomatous inflammation that subsequently leads to periportal fibrosis, portal hypertension, haemorrhage or even death. Macrophages are critical for granuloma formation and the development of liver fibrosis during schistosomiasis. However, whether the aberrant regulation of macrophage autophagy has an effect on the development of liver immunopathology in schistosomiasis remains to be elucidated. In this study, we showed that Schistosoma japonicum (S. japonicum) egg antigen (SEA)‐triggered macrophage autophagy limited the development of pathology in host liver. However, engagement of IL‐7 receptor (IL‐7R/CD127) on macrophages by S. japonicum infection‐induced IL‐7 significantly suppressed SEA‐triggered macrophage autophagy, which led to an enhanced liver pathology. In addition, anti‐IL‐7 neutralizing antibody or anti‐CD127 blocking antibody treatment increased macrophage autophagy and suppressed liver pathology. Finally, we demonstrated that IL‐7 protects macrophage against SEA‐induced autophagy through activation of AMP‐activated protein kinase (AMPK). Our study reveals a novel role for IL‐7 in macrophage autophagy and identifies AMPK as a novel downstream mediator of IL‐7‐IL‐7R signalling and suggests that manipulation of macrophage autophagy by targeting IL‐7‐IL‐7R signalling may have the potential to lead to improved treatment options for liver pathogenesis in schistosomiasis.

mediate collagen synthesis through a variety of mechanisms and finally result in periportal fibrosis, portal hypertension, haemorrhage or even death. 2,7,8 Autophagy of the macrophages has a critical regulatory function in down-regulation of liver immunopathology in various diseases. For example, macrophage autophagy functions to limit liver inflammation in D-galactosamine/lipopolysaccharide (GalN/LPS)-treated mice by inhibiting the generation of inflammasome-dependent IL-1b. 9 In high-fat diet-fed, LPS-treated mice, the loss of macrophage autophagy promoted liver inflammation. 10 Macrophage autophagy deficiency not only enhances infiltration of the liver by inflammatory cells, but exacerbates liver fibrosis by enhancing the fibrogenic properties of hepatic myofibroblasts via an IL-1-dependent pathway in mice exposed to CCl4. 11 However, given that macrophage plays critical role in schistosome egg-triggered immunopathology in host liver, little is known about whether and how aberrant regulation of macrophage autophagy can impact the liver immunopathology after schistosome infection.
Pro-inflammatory cytokine IL-7 is a "stromal cytokine" produced in the liver, bone marrow, lymph node, skin and gut. 12-14 IL-7 exerts a variety of effects by binding to the IL-7 receptor (IL-7R), consisting of IL-7Ra (CD127) and cc (CD132). 14 Although the underline mechanisms are still unknown, the role of IL-7 in modulating immunopathology during schistosome infection has been reported more than a dozen years ago. IL-7 deficiency markedly reduced level of collagen in the livers of S. mansoni-infected mice. 15 Reversely, S. mansoni-infected IL-7 transgenic mice showed aggravated collagen deposition. 16 Recently, study suggests that IL-7 may be involved in the autophagic process. In a T-cell line, D1, deprivation of IL-7 caused an increased number of autophagosomes, suggesting a possible antiautophagic role of IL-7 in T cells. 17 However, whether IL-7 can also regulate macrophage autophagy and subsequently impact on the tissue immunopathology has never been reported.
In this study, we demonstrated that IL-7 suppressed macrophage autophagy and enhanced liver immunopathology in S. japonicuminfected mice. The effect of IL-7 on liver immunopathology was mediated by the inhibition of AMP-activated protein kinase (AMPK)dependent macrophage autophagy. Our results suggest that induction of macrophage autophagy through IL-7 blockade may be a potential therapeutic strategy against tissue immunopathology in diseases including schistosomiasis.

| Ethics statement
Animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1), and all efforts were made to minimize suffering. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University for the use of laboratory animals (Permit Number: NJMU 20121110).

| Cytokine and antibody treatment of infected mice
Three independent experiments were carried out. In each experiment, 36 S. japonicum-infected mice were randomly assigned in six groups (six mice per group) to receive injections of either cytokine, antibody or their control solutions 3 weeks after S. japonicum infection. Mice were killed for further experiment 6 weeks after infection.

| Preparation of liver macrophages for analyses of CD127 expression
Perfused mice liver tissue was homogenized, and non-parenchymal cell pellets were obtained. Then hepatic mononuclear cells (MNCs) were isolated by using Percoll density-gradient centrifugation.

| Purification of peritoneal macrophages (PMΦs) and in vitro treatment
PMΦs were widely used as substitute for liver macrophages to obtain sufficient number of pure macrophages. 18,19 Briefly, peritoneal exudate cells were obtained from normal mice by peritoneal lavage, and PMΦs were purified from peritoneal exudate cells by adherence. The purity of enriched macrophages was >99%. Purified PMΦs were treated with PBS, 10 lg/mL SEA, 10 ng/mL IL-7 or 10 lg/mL SEA plus 10 ng/mL IL-7 for 24 hours.      and stained with H&E or Sirius Red. To perform granuloma area measurements, at least 100 granulomas with a single well-defined egg were randomly chosen from each group at 209 objective lens, and the areas were measured using Zeiss AxioVision software (Zeiss, Oberkochen, Germany). In addition, slides stained with Sirius Red were used to measure the intensity of fibrosis. At least 10 digital images (1009) were captured from slides of each mouse. Captured images were quantified using Image-Pro Plus system (Media Cybernetics, Silver Spring, MD). A total fibrosis density score was determined by dividing the image intensity by the image area. Intensity exclusion parameters were identical for each of the images captured.

| 3355
Autophagosomes were quantified on the basis of three independent experiments. In each experiment, 50 macrophages from each group were randomly selected for analysis.

| Statistical analysis
Statistical comparisons between different treatments were performed by an unpaired Student's t test, and P ≤ .05 was considered statistically significant. P values are as indicated by asterisks: *P ≤ .05, **P ≤ .01, ***P ≤ .001. 3.2 | IL-7-IL-7R signalling suppresses macrophage autophagy in S. japonicum-infected mice For the first time, our results showed that the specific chain of IL-7R (CD127) was expressed in macrophages (Figure 2A,B). To determine the role of IL-7-IL-7R signalling in macrophage autophagy in S. japonicum-infected mice liver, IL-7, anti-IL-7 neutralizing antibody or anti-CD127 blocking antibody were injected into mice F I G U R E 2 IL-7-IL-7R signalling suppresses macrophage autophagy in Schistosoma japonicum-infected mice. A, Total RNAs of RAW 264.7, BV-2, purified peritoneal macrophages (PMΦs) from normal mice, and FACS sorted liver macrophages from normal mice were analysed for Cd127 mRNA expression by RT-PCR followed by agarose gel electrophoresis. B, Expression of CD127 was measured by FCM. Grey-filled lines in FCM plots indicate the isotype control, and unfilled lines indicate the CD127-stained cells. Agarose gel and FCM plots are representative of three independent experiments. S. japonicum-infected mice were injected with PBS, IL-7, goat IgG isotype antibody, anti-IL-7 neutralizing antibody, rat IgG isotype antibody or anti-CD127 blocking antibody as described in Methods. Autophagosomes in macrophages of liver tissue were detected by TEM. C, Images were taken at either 12 0009 or 40 0009. The 40 0009 image is the enlarged image in the black frame.

| IL-7 suppresses macrophage autophagy via AMPK
Besides the role of AMPK as a master regulator of cellular metabolism, emerging evidence implicates its role in the prevention of autophagy. 24,25 Our results in Figure 5A,B showed that SEA significantly suppressed, while IL-7 significantly induced the activation of AMPK in purified PMΦs. Metformin (Met) and compound C were widely used as pharmacological activator or inhibitor of AMPK, respectively. 26,27 Results showed that Met significantly decreased levels of LC3II expression ( Figure 5C,D) and autophagosomes ( Figures 5E and S4) in all groups, including the significant decrease in the pro-autophagic effect of SEA and the enhancement of the anti-autophagic effect of IL-7. However, compound C significantly increased levels of LC3II expression ( Figure 5F,G) and autophagosomes ( Figures 5H and S5) in all groups. These results suggested that SEA and IL-7 might promote or prevent macrophage autophagy by inhibiting and stimulating AMPK activation, respectively.
F I G U R E 5 IL-7 suppresses macrophage autophagy via AMP-activated protein kinase (AMPK). Purified PMΦs from normal mice were treated as described in Figure 3A  To further confirm that AMPK is required for the anti-autophagic effects of IL-7, expression of AMPK was down-modulated using a-subunit-targeted siRNA. Immunoblot analysis revealed that siRNA targeting a-subunit dramatically reduced the total and phosphorylated AMPKa levels ( Figure 6A). Meanwhile, siRNA treatment also significantly increased LC3II expression ( Figure 6B,C) and autophagosomes ( Figures 6D and S6) in all groups. Taken together, these results indicate that IL-7 protects macrophages from autophagy via AMPK.

| DISCUSSION
In hosts infected with S. japonicum or mansoni, parasite eggs are trapped in hosts' liver and induce severe liver granulomatous responses, which subsequently lead to liver fibrosis, circulatory impairment or even death. 2,3 Macrophage is one of the critical participants in development of the liver immunopathology initiated by parasite eggs in host liver. 2,8 In this study, we showed for the first time that IL-7 induced in S. japonicum infection suppressed AMPKdependent macrophage autophagy and enhanced liver immunopathology.
Consistent with previous study, 28  Our data showed that macrophage apoptosis was inhibited by the autophagy inhibitor 3-MA. This result is in line with previous studies.
For example, in hepatoma cells, inhibition of autophagy via BafA 1 or Atg5 siRNA decreases ASPP2 (apoptosis-stimulating protein of p53-2)-induced apoptosis. 20 In primary cortical neurons, inhibition of autophagy by 3-MA, or lentivirally delivered shRNAs against Atg5 and Atg7, strongly reduced the staurosporine-induced apoptosis. 21 As macrophage is a major component of granuloma cells, macrophage autophagy might functions to limit liver pathology by its pro-apoptotic activity. And, we have demonstrated that IL-7 suppresses macrophage autophagy and exacerbated liver pathology simultaneously during S. japonicum infection, and we speculate that IL-7 might enhance liver immunopathology through a mechanism involving inhibition of the autophagic apoptosis in macrophage. Meanwhile, we also understand that except for by suppressing autophagy of macrophages in liver, IL-7 may also contribute to the regulation of the liver immunopathology by its anti-apoptotic effect on T cells.
To date, mechanisms by which IL-7 regulates autophagy remain poorly investigated. In this study, we identified AMPK as a novel target downstream of IL-7 signalling to suppress macrophage autophagy. We postulate that AMPK inhibits autophagy via inactivation of the mTOR pathway through TSC1/2-complex activation, based on the finding that mTOR promotes autophagy by inhibition of Akt, a well-known inhibitor of autophagy, in human cancer cells. 29 However, more studies need to be done in the future to further elucidate details of the mechanisms involved in the regulation of AMPK activation by IL-7.
To sum up, our study reports a novel role of IL-7 in regulation of macrophage autopahgy and identifies AMPK as a downstream target of IL-7-IL-7R signalling, and suggests manipulation of macrophage autophagy through IL-7-IL-7R pathway as a possible therapeutic option for schistosomiasis.