B1a cells protect against Schistosoma japonicum–induced liver inflammation and fibrosis by controlling monocyte infiltration

During Schistosoma infection, lack of B cells results in more severe granulomas, inflammation, and fibrosis in the liver, but the mechanisms underlying this pathology remain unclear. Thus, our aim was to clarify the mechanisms underpinning the immunomodulation of B cells in mice infected with Schistosoma japonicum. We found that B cell deficiency led to aggravated liver pathology, as demonstrated by increases in the size of the egg-associated granulomas, alanine transaminase levels, and collagen deposition. Compared with infected wild-type mice, infected B cell–deficient μMT mice showed increased infiltration of Ly6Chi monocytes and higher levels of proinflammatory cytokines (tumor necrosis factor alpha, interleukin 6, and interleukin 12) and chemokines ([C-C motif] ligands (CCL)2, CCL3, CCL4, and CCL5). The results of flow cytometric analysis and cell transfer experiments showed that B1a cells increased significantly in the liver following S. japonicum infection, with some of those cells deriving from the peritoneal cavity. We also found that secretion of IL-10 from hepatic B cells increased significantly in infected wild-type mice and that this IL-10 was mainly derived from B1a cells. In addition, adoptively transferring peritoneal cavity B cells purified from wild-type, but not from IL-10–deficient mice, to μMT mice significantly reduced liver pathology and liver infiltration of Ly6Chi monocytes. These reductions were accompanied by decreases in the expression levels of chemokines and inflammatory cytokines. Taken together, these data indicated that after S. japonicum infection, an increased number of hepatic B1a cells secrete IL-10, which inhibits the expression of chemokines and cytokines and suppresses the infiltration of Ly6Chi monocytes into the liver thereby alleviating liver early inflammation and late fibrosis. Understanding this immunomodulatory role of B1a cells in schistosomiasis may lead to the development of therapeutic strategies for Schistosoma-induced liver disease. Author summary Infection with Schistosoma, a waterborne parasitic flatworm (trematode) commonly called a blood fluke, results in strong granulomatous inflammation caused by the deposition of eggs in the liver. A granuloma is a substantial immune cell infiltration around the eggs intermixed with liver cells that can protect the host against liver damage. However, excessive infiltration and inflammation can lead to severe liver injury and fibrosis. Here, we found that B1a cells accumulate in the liver of mice after S. japonicum–induced infection and that these B1a cells release the anti-inflammatory cytokine interleukin 10 to regulate inflammation. The B1a cell–derived interleukin 10 inhibits the expression of chemokines (which attract cells such as monocytes to sites of infection or inflammation) and thus restrains excessive infiltration of Ly6Chi monocytes (which may have proinflammatory activity) into the liver, thereby alleviating early inflammation and later fibrosis. Our study provides insight into the immunomodulation of B1a cells in schistosomiasis and offers key information for the development of therapeutic strategies in Schistosoma-induced liver disease.

showed no significant changes between WT mice and μMT mice (Fig 2). MoMFs [24]. In the present study, we found that a larger number of proinflammatory 132 Ly6C hi monocytes infiltrated the liver in μMT mice than in WT mice 6 weeks after 133 infection. By contrast, the numbers of Kupffer cells and Ly6C lo MoMFs were similar 134 in μMT mice and WT mice (Fig 3). 135 We hypothesized that the increased macrophage number in the liver of μMT mice 136 after infection reflects either enhanced monocyte production or increased monocyte 137 recruitment. Because Ly6C hi monocytes are derived from the bone marrow and 138 circulate in the blood [25], we analyzed Ly6C hi monocytes in the peripheral blood. We higher in the liver of μMT mice than in WT mice ( Fig 4A). We also detected the protein 147 levels of some key chemokines. The protein levels of CCL2, CCL3,CCL4,and CCL5 148 in the liver of μMT mice were significantly increased compared with those of WT mice 149 ( Fig 4B). These data suggest that mobilization and recruitment, rather than production, 150 accounted for differences in monocyte infiltration. Therefore, B cells limit monocyte

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To investigate the role and mechanism of B cell action in this murine model, we 161 first determined the number of total B cells in the liver during infection. We found that 162 the B cell number was significantly increased 6 weeks after infection in WT mice (Fig   163   5A). Further analysis of hepatic B cell subsets showed that both the percentage and 164 number of hepatic B1a cells were markedly increased 6 weeks after infection, and the 9 165 numbers of B1b cells and B2 cells were also increased (Fig 5B,5C,and S4 Fig). In 166 addition, we found that both the percentage and number of PC B1a cells were markedly 167 decreased 6 weeks after infection, whereas the percentage and number of PC B2 cells 168 were increased (Fig 5D and E). These data suggest that the increased B1 cells in the 169 liver after infection were recruited from the PC. To provide further support for this 170 finding, we conducted adoptive cell transfer experiments. B cell-deficient μMT mice 171 were infected with S. japonicum and then were intraperitoneally injected with 172 uninfected WT mice-derived PC B cells or phosphate-buffered saline (PBS) 4 weeks 173 after infection. Samples were harvested 6 weeks after infection ( Fig 6A). The purity of 174 WT mice-derived PC B cells was more than 95% ( Fig 6B). After the cell transfer, B 175 cell subsets could be detected in the PC and liver of μMT mice. Compared with those 176 in donor WT mice, the percentage of B1a cells in the PC was lower in the recipient 177 μMT mice, whereas the percentage of B1a cells in the liver was higher, which was 178 consist with our observations in the infected WT mice (Fig 6C-F). induced liver pathology via IL-10, we first examined IL-10 expression in the liver and 184 B cells. The hepatic IL-10 protein levels in μMT mice were significantly lower than 185 those of WT mice 6 weeks after infection (Fig 7B), suggesting that B cells contribute 186 to IL-10 production in the liver after infection. Our data also showed that IL-10 expression levels in B cells were increased after infection (Fig 7C, D), especially in 188 hepatic B1a cells (Fig 7E).

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Infiltrating Ly6C hi monocytes may act as a double-edged sword in liver damage.

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These cells express a substantial number of inflammatory cytokines and chemokines 225 and promote liver inflammation, injury, and fibrosis in the initiation and progression of 226 various types of liver injury, including acute viral injection, hepatotoxicity following 227 CCl 4 treatment, or ischemia-reperfusion damage [24,33,34]. We hypothesized that 228 recruited Ly6C hi monocytes also contribute to the initial liver damage and development 229 of fibrosis after S. japonicum infection. As expected, S. japonicum-infected μMT mice 230 with increased Ly6C hi monocyte infiltration had higher levels of ALT and liver fibrosis than WT mice (Fig 1 and 3). When liver injury ceases, inflammatory Ly6C hi monocytes 232 mature into Ly6C lo restorative macrophages, which display increased expression of cavities migrate to neighboring lymphoid sites or tissues [19,39]. In the present study, we found that B1 cells, especially B1a cells, migrated from the PC to the liver after S. 254 japonicum infection, which was shown by the increased percentage and number of B1a 255 cells in the liver and their concurrent decrease in the PC after infection (Fig 5). In expressing IL-10 in the S. japonicum-infected liver (Fig 7). We also found that in the 274 absence of IL-10, the transferred PC B cells were unable to downregulate granuloma inflammation, recruitment of monocytes, or the expression of a number of 276 proinflammatory chemokines and cytokines in the infected μMT mice (Fig 8). These 277 data suggest that after S. japonicum infection, B cells control the recruitment of 278 monocytes and the expression of proinflammatory chemokines and cytokines via IL-10 279 production.

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The cross talk between B cells and monocytes observed in our study appears to be 281 opposite to that observed in CCl 4 -induced fibrosis [31]. The difference may be that 282 different liver microenvironments induce different B cell subsets in these two models.

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In the CCl 4 model, the increased B cells in the liver produce IgG and express CD138,  Biosciences) with FlowJo (version 7.6.1) software.

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Total hepatic RNA was isolated from frozen liver tissue using Trizol (Invitrogen).

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The MACS-sorted hepatic B cells were resuspended in Trizol, and the RNA was 356 isolated according to the manufacturer's instructions. First strand cDNA was 357 synthesized from ≤500 ng of RNA using a PrimeScript RT reagent kit (TaKaRa).

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All data are expressed as mean ± SD and were analyzed using GraphPad Prism 383 6.01 software. Two-tailed, unpaired Student's t tests were used to compare variables 384 between two groups. P < 0.05 was considered statistically significant.