Kidney Macrophages Research Articles

TcpC Inhibits M1 but Promotes M2 Macrophage Polarization via Regulation of the MAPK/NF-κB and Akt/STAT6 Pathways in Urinary Tract Infection.

TcpC is a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC). Macrophages can differentiate into two different subsets M1 and M2 that play distinct roles in anti-infection immunity. Here, we investigate the influence of TcpC on M1/M2 polarization and the potential mechanisms. Our data showed that M1 markers CD86 and iNOS were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced in macrophages in kidneys from the TcpC-secreting wild-type CFT073 (CFT073wt)-infected pyelonephritis mouse model, compared with those in macrophages in kidneys from TcpC knockout CFT073 mutant (CFT073Δtcpc)-infected mice. CFT073wt or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in both human and mouse macrophage cell lines THP-1 and J774A.1. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but enhanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.Simple SummaryWe investigate the influence of TcpC, a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC), on M1/M2 macrophage polarization and the potential mechanisms. TcpC-secreting wild-type CFT073 (CFT073wt) or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in CFT073wt-infected pyelonephritis model mouse and both human and mouse macrophage cell lines THP-1 and J774A.1, respectively. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but en-hanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.

Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus

Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a key regulator of inflammatory responses by scavenging proinflammatory extracellular ATP (eATP) and promoting the generation of immunosuppressive adenosine. In addition to the previously identified CD39 homolog (designated CD39a), here we identified two novel functional CD39 splice variants (termed CD39b and CD39c) from teleost fish Japanese flounder (Paralichthys olivaceus). Transcriptional expression analysis revealed that all three CD39 splicing variants were coexpressed in Japanese flounder head kidney macrophages (HKMs) and a variety of normal tissues but with differential tissue/immune cell expression preferences. Immune challenge experiments showed that the expression of CD39 splice variants was modulated by both in vitro inflammatory stimulation and in vivo bacterium Edwardsiella piscicida infections. However, even though all CD39 splice variants were functional cell surface-expressed proteins and shared generally similar domain structures, their varied cytoplasmic N-terminal sequences resulted in a varied capability for hydrolysis of eATP and attenuation of eATP/P2XR-mediated proinflammatory cytokine expression. Pharmacological inhibition of endogenous CD39 activity in Japanese flounder HKMs or overexpression of CD39 splice variants in Japanese flounder FG-9307 cells further revealed the role of CD39 variants in down-regulating LPS-induced proinflammatory cytokine production. In addition, luciferase reporter analysis demonstrated that the CD39 splice variant promoters had varied transcriptional activities and that Sp1 was a potential transcription factor for CD39c transcription regulation. Taken together, we report for the first time that functional CD39 splice variants provide additional means to downregulate eATP- and LPS-induced proinflammatory response(s) in fish.

Usability of Immortalized Porcine Kidney Macrophage Cultures for the Isolation of ASFV without Affecting Virulence.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

NGAL release from peripheral blood mononuclear cells protects against acute kidney injury and prevents AKI induced fibrosis

We propose the use of a peripheral blood mononuclear cell therapy based on cell NGAL release to be used in the clinical setting for acute kidney injury (AKI) and the derived fibrosis. First, we designed a procedure whereby PBMC overexpress NGAL and anti-inflammatory agents when subjected to repetitive anoxia/reoxygenation (PBMC (A/R)). Using an in vivo AKI model, we observed that PBMC(A/R) reduces BUN and creatinine levels in blood and inflammation, enhances anti-inflammation, induces proliferation of tubular epithelial cells and reduces AKI-induced fibrosis. Flow cytometry analysis evidenced that monocytes are the only cells accumulated in the injured kidney and phenotype analysis of freshly isolated kidney macrophages, revealed that the healing phenotype is maintained the time needed for recovery. NGAL release from PBMC(A/R) determines the beneficial effect of the therapy since administration of a NGAL antibody previous to the therapy or injection of PBMC(A/R) obtained from NGAL KO animals abolished the beneficial effects. CD11b–NGAL positive cells were enhanced in tissue after PBMC (A/R) therapy and were produced by the injected monocytes. In an in vitro model with tubular epithelial cells (NRK52e) we proved that NGAL release by PBMC(A/R) induced epithelial proliferation and activation of PI3K/Akt pathway.

Immunopotentiation mechanism of a mannose-modified peptide fragment containing a dominant antigenic epitope of Micropterus salmoides rhabdovirus (MSRV) glycoprotein after coupling to nanocarrier

Micropterus salmoides rhabavirus (MSRV) is an important pathogen that threatens the aquaculture of bass farming worldwide, and the efficacy of vaccines developed against this pathogen still needs to be improved. In order to further enhance the immune efficacy of existing vaccines, a mannose-modified nanovector vaccine SWCNTs-M-G2 was constructed by screening for peptide fragments encoding highly immunogenic antigenic epitopes (G2) and mannose modifications. Mannose receptor (MR) is abundantly expressed on the surface of Antigen-presenting cells (APCs) and plays a role in recognizing pathogens and presenting antigens. In this study, the effect of mannosylated modification on the immunopotentiation of highly immunogenic peptide fragments was investigated by using the characteristics of MR-specific recognition of mannose and APCs surface. In vitro macrophage assays showed that SWCNTs-M-G2 was efficiently phagocytosed and taken up by largemouth black bass head kidney macrophages, and effectively stimulated macrophage activation and upregulated the secretion of macrophage cytokines IL-1β, IL-8 and IL-12. In vivo, SWCNTs-M-G2 was also shown to be better taken up by immune organs such as spleen and head kidney, and induced increased expression of IL-1β, IL-8 and IL-12 in the immune organs of Micropterus salmoides, and promoted the expression of TCR and CD4 molecules in specific immune cells. Meanwhile, in vivo fluorescence imaging revealed that SWCNTs-M-G2 could be in the immune organ of fish for a longer period of time, contributing to the efficient uptake of antigens. Finally, the challenge test showed that SWCNTs-M-G2 could mediate the production of specific antibodies at a higher level than the unmodified carrier vaccine SWCNTs-G2 and showed a higher relative immune protection rate.

Heat acclimation ameliorated heat stress-induced acute kidney injury and prevented changes in kidney macrophages and fibrosis.

Heatstroke can cause acute kidney injury (AKI), which reportedly progresses to chronic kidney disease. Kidney macrophages may be involved in such injury. Although heat acclimation (HA) provides thermal resilience, its renoprotective effect and mechanism remain unclear. To investigate heat stress-induced kidney injuries in mice and the mitigating effect of HA on them, male C57/BL6J mice were exposed to heat stress (40°C, 1 h) with or without 5-day HA (38°C, 3 h/day) prior to heat stress. Heat stress damaged kidney proximal tubules with an elevation of urinary kidney injury molecule-1. Kidney fibrosis was observed on day 7 and correlated with urinary kidney injury molecule-1 levels on day 3. Kidney resident macrophages decreased on day 1, whereas the number of infiltrating macrophages in the kidney did not change. Both subsets of macrophages polarized to the proinflammatory M1 phenotype on day 1; however, they polarized to the anti-inflammatory M2 phenotype on day 7. HA significantly ameliorated heat stress-induced proximal tubular damage and kidney fibrosis. HA substantially increased heat shock protein 70 expression in the tubules before heat stress and reduced the elevation of cleaved caspase-3 expression after heat stress. HA also induced heat shock protein 70 expression of resident macrophages and prevented heat stress-induced changes in both subsets of kidney macrophages. These results provide pathophysiological data supporting the renoprotective effect of HA. Further studies are needed to confirm that HA can prevent kidney damage due to heat stress in humans.NEW & NOTEWORTHY Heat stress could induce acute kidney injury. Although heat acclimation (HA) reportedly provides thermal tolerance, its effect on heat stress-induced kidney damage remains unclear. This study showed that 5-day HA ameliorates mouse kidney tubular damage and subsequent fibrosis caused by heat stress. It also demonstrated that HA enhances intracellular heat shock protein 70 expression in tubular cells and prevents a decrease in kidney resident macrophages, which explains the renoprotective effect of HA.

DsDNA-induced AIM2 pyroptosis halts aberrant inflammation during rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a severe condition that commonly leads to acute kidney injury (AKI). While double-stranded DNA (dsDNA) released from injured muscle can be involved in its pathogenesis, the exact mechanism of how dsDNA contributes to rhabdomyolysis-induced AKI (RIAKI) remains obscure. A dsDNA sensor, absent in melanoma 2 (AIM2), forms an inflammasome and induces gasdermin D (GSDMD) cleavage resulting in inflammatory cell death known as pyroptosis. In this study using a mouse model of RIAKI, we found that Aim2-deficiency led to massive macrophage accumulation resulting in delayed functional recovery and perpetuating fibrosis in the kidney. While Aim2-deficiency compromised RIAKI-induced kidney macrophage pyroptosis, it unexpectedly accelerated aberrant inflammation as demonstrated by CXCR3+CD206+ macrophage accumulation and activation of TBK1-IRF3/NF-κB. Kidney macrophages with intact AIM2 underwent swift pyroptosis without IL-1β release in response to dsDNA. On the other hand, dsDNA-induced Aim2-deficient macrophages escaped from swift pyroptotic elimination and instead engaged STING-TBK1-IRF3/NF-κB signalling, leading to aggravated inflammatory phenotypes. Collectively, these findings shed light on a hitherto unknown immunoregulatory function of macrophage pyroptosis. dsDNA-induced rapid macrophage cell death potentially serves as an anti-inflammatory program and determines the healing process of RIAKI.

Abstract LB051: IL-6 and CCL18 mediate reciprocal interactions between VHL-deficient kidney cells and macrophages during development of ccRCC

Abstract Loss of the von Hippel-Lindau (VHL) tumor suppressor gene function accounts for 70-80% of all clear-cell renal cell carcinoma (ccRCC) cases, the most prevalent form of RCC. Accumulating evidence has indicated that ccRCC arises at the site of chronic inflammation; however, how the ccRCC tumor cells interact with the immune components of the microenvironment has remained unclear. In this study we employed unbiased proteomic and genomic analyses on components of the tumor microenvironment under different conditions. We have identified the molecular and cellular mechanisms that underlie the crosstalk between VHL-deficient kidney tubule cells and macrophages with relevance to tumor-associated inflammation and ccRCC development. We demonstrate that the VHL-deficient non-cancerous kidney epithelial cells, representing the early stage of ccRCC initiation, secrete IL-6 that recruits macrophages and induces their polarization toward the pro-tumorigenic M2 phenotype in vitro and in the mouse Vhlh gene conditional knockout model. The activated human macrophages reciprocally secrete CCL18 and TGF-beta1 to stimulate epithelial-to-mesenchymal transition (EMT) of the kidney tubule cells. Importantly, treatment with anti-IL-6 neutralizing antibody can rescue inflammatory, proliferative, and EMT phenotypes of Vhlh conditional knockout in vivo. Furthermore, in a human ccRCC xenograft model, exogenous human macrophages can significantly promote primary tumor growth and metastasis; and this oncogenic function is dependent on the expression of CCL18. These findings identify specific factors involved in a reciprocal crosstalk between the tumor cells and the immune components such as macrophages in the microenvironment. These results thus suggest an avenue for early intervention of ccRCC. Citation Format: Tien Hsu, Thi Ngoc Nguyen, Hieu-Huy Nguyen-Tran, Chen-Yun Chen. IL-6 and CCL18 mediate reciprocal interactions between VHL-deficient kidney cells and macrophages during development of ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB051.

IL6 and CCL18 Mediate Cross-talk between VHL-Deficient Kidney Cells and Macrophages during Development of Renal Cell Carcinoma.

The identification of VHL-deficient kidney tubule cell cross-talk with macrophages regulated by IL6 and CCL18 reveals potential targets for the prevention and treatment of ccRCC.

Macrophage migration inhibitory factor (MIF) of golden pompano (Trachinotus ovatus) is involved in the antibacterial immune response

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%–94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.

MO285: FOSL1 Preserves Klotho Expression and Limits Acute Kidney Injury

Abstract BACKGROUND AND AIMS Fosl1 (FRA1, Fos-related-antigen 1) is a transcription factor of the AP-1 complex (activator protein 1) that regulates the expression of genes that regulates processes involved in acute kidney injury (AKI) such differentiation, inflammation, proliferation and cell death. We have now explored the role of Fosl1 in AKI and repair. METHOD The function of Fosl1 was explored in murine nephrotoxic AKI induced by folic acid or cisplatin in wild type and genetically modified mice tubular cell Fosl1 deficient mice (Fosl1Δtub). RT-qPCR, western blot, and immunohistochemistry were used to assess gene and protein expression. Direct regulation of Klotho by Fosl1 was explored by chromatin immunoprecipitation (ChIP). Murine tubular cells were cultured in an inflammatory milieu promoted by TWEAK and Fosl1 was targeted by siRNA. RESULTS Fosl1 was one of the most upregulated transcription factors in experimental nephrotoxic AKI transcriptomics and also one the transcription factors binding to the Klotho promoter (Panel A). Fosl1 upregulation was confirmed in experimental nephrotoxic AKI and in human AKI and localized to tubular cell nuclei at early times points (panel B). Fosl1 deficient (Fosl1Δtub) mice developed more severe folic acid- or cisplatin-induced AKI and higher proinflammatory cytokine expression and kidney macrophage infiltration (Panel C). Additionally, kidney expression of the nephroprotective and antiaging factor Klotho was more severely depressed in Fosl1Δtub mice (Panel D). Enrichment of AP1 binding sites was found in the Klotho promoter and we confirmed direct Fosl1 binding to the Klotho promoter in a ChIP assay. Moreover, Fosl1Δtub mice had disturbed expression of cell cycle regulators and PCNA expression, suggesting a dysregulation in kidney repair. In tubular cells cultured in an inflammatory milieu, Fosl1 expression was also upregulated. Fosl1 siRNA targeting in cultured tubular cells increased inflammatory gene expression and cell death and decreased Klotho expression. CONCLUSION Fosl1 contributes to an adaptive kidney response during AKI that limits kidney injury and includes Klotho expression and Fosl1 deficiency promotes an accelerated loss of klotho and more severe AKI.

The analysis of the effects of propolis products as food supplements on the viability of baby hamster kidney (BHK-21) and murine macrophage (RAW 264.7) cells by spectrophotometric MTT assay

Propolis is beeswax with rich bioactive compound content. On the market, there many propolis products as food supplements for the consumers. This study aimed to investigate the effect of food supplement products based on propolis at several concentrations on the viability of baby hamster kidney cells and murine macrophage cell lines (BHK-21 and RAW 264.7). For this purpose, both cell lines were treated with the two-fold serial dilutions (from 20 to 2-10) of each six propolis products (P1, P2, P3, P4, P5 and P6) after reaching monolayer cell in 96-well microplates. The viability and inhibition of cells were spectrophotometrically determined by MTT assay after 24 h. For BHK-21, the IC50s of P1, P2, P3, P4, P5 and P6 were calculated as 0.003, 0.178, 0,082, 0.451, 0.278 and 0.384 %, respectively. For RAW 264.7, the IC50 of P1, P2, P3, P4, P5 and P6 were calculated as 0.260, 0.218, 0.115, 0.257, 0.207 and 0.265%, respectively. The IC50 value was higher for RAW 264.7 cells than for BHK-21 cells. So, the low cytotoxic effect was determined in RAW 264.7 cells. Propolis products contain some additives (aroma, flavouring) had lower the IC50 and the lower viability of BHK-21 cells. So, Additives in the propolis food supplement might be an effective factor on cell viability as much as dilution factor and propolis content.

Characterization, evolution and expression analysis of Toll-like receptor 7 (TLR7) in turbot (Scophthalmus maximus L.)

The pattern recognition receptors (PRRs) can recognize the conserved molecular structures of pathogens to active the innate immune responses, and subsequently induce the antigen-specific adaptive immune responses for the clearance of infected pathogen. Among the PRRs, Toll-like receptors (TLRs) are the first and best characterized PRRs across all the species. Among the TLR members, TLR7 showed significant conservation across the vertebrates, with the lowest rate of evolution for its LRR domains from primates to fishes. In the current study, one TLR7 (SmTLR7) gene was captured in turbot, with a 3144 bp open reading frame (ORF), that encoding 1047 amino acid residues. Following multiple sequence comparison, SmTLR7 was found to have the highest similarity and identity both to Paralichthys olivaceus with 91.9% and 85.9%, respectively. In phylogenetic analysis, SmTLR7 was firstly clustered with Japanese flounder, and then clustered with fugu, rainbow trout, and zebrafish. In addition, SmTLR7 was widely expressed in all the examined tissues with the highest expression level in spleen, followed by skin, while the lowest expression level was detected in blood. Following both Edwardsiella tarda and Vibrio anguillarum challenge, SmTLR7 was significantly down-regulated in gill and intestine, and up-regulated in skin. Moreover, SmTLR7 was significantly up-regulated in head kidney macrophages following LPS, LTA, PGN and polyI:C stimulation, as well as showed the strongest binding ability to LPS, followed by PGN, LTA, and polyI:C in a dose-dependent manner. Finally, following RNAi of SmTLR7, MyD88 and IL-1β were slightly up-regulated, while TRAF6 and IL-8 were significantly down-regulated. The characterization of TLR7 can expand our understanding of the PRRs in teleost fishes, and eventually aid the exploration of interactions between host and pathogen.

Different Spatial and Temporal Roles of Monocytes and Monocyte-Derived Cells in the Pathogenesis of an Imiquimod Induced Lupus Model.

Mounting evidence indicates the importance of aberrant Toll-like receptor 7 (TLR7) signaling in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of disease progression remains unclear. An imiquimod (IMQ)-induced lupus model was used to analyze the lupus mechanism related to the aberrant TLR7 signals. C57BL/6 mice and NZB/NZW mice were treated with topical IMQ, and peripheral blood, draining lymph nodes, and kidneys were analyzed focusing on monocytes and monocyte-related cells. Monocytes expressed intermediate to high levels of TLR7, and the long-term application of IMQ increased Ly6Clo monocytes in the peripheral blood and Ly6Clo monocyte-like cells in the lymph nodes and kidneys, whereas Ly6Chi monocyte-like cell numbers were increased in lymph nodes. Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice were supplied by bone marrow-derived cells as demonstrated using a bone marrow chimera. Ly6Clo monocytes obtained from IMQ-induced lupus mice had upregulated adhesion molecule-related genes, and after adoptive transfer, they showed greater infiltration into the kidneys compared with controls. RNA-seq and post hoc PCR analyses revealed Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice had upregulated macrophage-related genes compared with peripheral blood Ly6Clo monocytes and downregulated genes compared with kidney macrophages (MF). Ly6Clo monocyte-like cells in the kidneys upregulated Il6 and chemoattracting genes including Ccl5 and Cxcl13. The higher expression of Il6 in Ly6Clo monocyte-like cells compared with MF suggested these cells were more inflammatory than MF. However, MF in IMQ-induced lupus mice were characterized by their high expression of Cxcl13. Genes of proinflammatory cytokines in Ly6Chi and Ly6Clo monocytes were upregulated by stimulation with IMQ but only Ly6Chi monocytes upregulated IFN-α genes upon stimulation with 2′3′-cyclic-GMP-AMP, an agonist of stimulator of interferon genes. Ly6Chi and Ly6Clo monocytes in IMQ-induced lupus mice had different features. Ly6Chi monocytes responded in the lymph nodes of locally stimulated sites and had a higher expression of IFN-α upon stimulation, whereas Ly6Clo monocytes were induced slowly and tended to infiltrate into the kidneys. Infiltrated monocytes in the kidneys likely followed a trajectory through inflammatory monocyte-like cells to MF, which were then involved in the development of nephritis.

Myeloid Piezo1 Deletion Protects Renal Fibrosis by Restraining Macrophage Infiltration and Activation.

Macrophages play important roles in renal fibrosis, partially by sensing mechanical forces, including shear stress and increased stiffness. The mechanically activated cationic channel Piezo1 drives vascular formation and blood pressure regulation to inflammatory responses, or cancer, but its role in macrophages in fibrotic kidney is elusive. Here, we hypothesized that Piezo1 in macrophages may have functions in renal fibrosis. We established a genetically engineered mouse model with Piezo1 specific knockout in myeloid cells and challenged with unilateral ureteric obstruction operation and folic acid treatment to induce the renal fibrosis, aiming to investigate the function of the mechanical-sensitive protein Piezo1 in macrophages in renal fibrosis and its underlying mechanisms. Myeloid Piezo1 was indispensable for renal fibrosis generation. Piezo1 gene deletion in the myeloid lineage was protective in mice with renal fibrosis. Further analyses revealed that macrophage accumulation in the injured kidney depended on the Piezo1-regulated C-C motif chemokine ligand 2, C-C motif chemokine receptor 2 pathway, and Notch signaling cascade. Moreover, Piezo1 deletion restrained macrophage inflammation and consequently suppressed kidney fibrosis and epithelial-mesenchymal transition. In vitro assays showed that Piezo1 deficiency blocked lipopolysaccharide and Piezo1 activation-induced inflammatory responses in bone marrow-derived macrophages. Mechanistically, Piezo1 regulated inflammation through the Ca2+-dependent intracellular cysteine protease, as the pharmacological inhibition of calpain blocked the proinflammatory role of Piezo1. This study characterized the important function of Piezo1 in renal fibrosis. Targeting the Piezo1 channels by genetic or pharmacological manipulations may be a promising strategy for the treatment of renal fibrosis.

Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Two immune-based methods using immortalized porcine kidney macrophages for quantifying neutralizing activity against porcine reproductive and respiratory syndrome virus-2

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a serious infectious disease in pigs in farms worldwide. Neutralizing antibody titer is an effective index for evaluating immunity to PRRSV; however, PRRSV has different neutralizing cross-reactivity between strains. Therefore, quantitative measurement of neutralizing antibody titers against field PRRSV strains would be required to evaluate whether neutralizing antibodies in pigs could possess neutralizing activity against individual or multiple strains. Immune-based methods, such as image cytometry (ICM) and cell-based enzyme-linked immune sorbent assay (ELISA), are quantitative and can be used to evaluate many samples. Using immortalized porcine kidney macrophages (IPKMs), which are highly susceptible to infection from field PRRSV-2 strains compared with other cell lines, immune-based methods could enable the evaluation of the neutralizing activity of porcine serum against field strains of PRRSV-2 that are difficult to isolate in conventional cells. In summary, we adapted two methods, namely ICM and cell-based ELISA, to IPKMs for quantitative neutralizing antibody titer measurements. Two immune-based methods using IPKMs are adequate for quantifying neutralizing activity of porcine serum against PRRSV-2, including field strains.

Assessment of the immune responses of macrophages in selective breeding pangasius families (Pangasianodon hypophthalmus) resistant to enteric septicemia of catfish (ESC)

This study aimed to evaluate the non-specific immune responses of macrophages in two selective breeding pangasius family groups: high disease-resistant (A) and low disease-resistant (B) to Enteric Septicemia of Catfish (ESC) caused by Edwardsiella ictaluri. Fish were sampled at five different time points after infection with the bacteria E. ictaluri including before the infection process, 24hpi, 48hpi, 264hpi and 312hpi. There were 372 samples including 192 samples of fish in group A and 180 samples in group B. All of them were used to analyze the amount of melano-macrophage centers in the tissues of liver, kidney and spleen; phagocytosis activity (PA) and phagocytosis index (PI) of the anterior kidney macrophages. Results showed that (1) the number of melano-macrophages of group A fish was higher than that of group B at survey points during the challenge and the difference was significant at 48hpi; (2) PA and PI of group A were higher than that of group B in the period from 48 to 264hpi, specifically, the PI of group A was higher than that of group B, which was statistically significant at 48hpi. The results of this study showed that the differences in non-specific immune responses through melano-macrophages can improve the survival rate and life span of the high disease-resistant family compared to the low disease-resistant family after the challenge with Edwardsiella ictaluri.

Dietary magnolol inclusion improves the antioxidant and immune responses, and resistance to Aeromonas hydrophila in genetically improved farmed tilapia (Oreochromis niloticus)

A 10-week feeding trial was conducted to evaluate the effects of dietary magnolol inclusion on the growth rate, antioxidant and immune responses of genetically improved farmed tilapia Oreochromis niloticus. Four experimental diets were formulated with 0%, 0.02%, 0.04% and 0.08% magnolol, respectively. Dietary 0.02–0.08% magnolol inclusion did not affect the growth rate and feed utilization of tilapia. However, dietary 0.02–0.08% magnolol inclusion increased the plasma catalase, the intestinal alkaline phosphatase (ALP), and the hepatic glutathione reductase (GR) and catalase activities; but reduced the hepatic malondialdehyde (MDA) content. Further, supplementation with 0.04–0.08% magnolol increased the plasma superoxide dismutase (SOD), peroxidase, ALP and total antioxidant capacity (TAC), the intestinal GR, catalase, peroxidase and TAC, and the hepatic SOD and TAC activities; but decreased the plasma MDA, the intestinal MDA and reactive oxygen species contents. Dietary 0.02–0.04% magnolol inclusion increased the plasma complement 3 content, whereas the plasma immunoglobulin M, complement 4 contents and lysozyme activity were increased by dietary 0.04–0.08% magnolol inclusion. The phagocytic activity of head kidney macrophages was increased by the inclusion of 0.04% magnolol. Additionally, supplementation with 0.02–0.08% magnolol increased the relative percentage survival of tilapia after infection with Aeromonas hydrophila. These results indicated that 0.04% magnolol inclusion improved the antioxidant and immune responses without compromising growth performance of tilapia, suggesting that magnolol could be used as an antioxidant and immunostimulant in aquafeed.

Kidney VISTA prevents IFN-γ/IL-9 axis-mediated tubulointerstitial fibrosis after acute glomerular injury.

Severe glomerular injury ultimately leads to tubulointerstitial fibrosis that determines patient outcome, but the immunological molecules connecting these processes remain undetermined. The present study addressed whether V-domain Ig suppressor of T cell activation (VISTA), constitutively expressed in kidney macrophages, plays a protective role in tubulointerstitial fibrotic transformation after acute antibody-mediated glomerulonephritis. After acute glomerular injury using nephrotoxic serum, tubules in the VISTA-deficient (Vsir–/–) kidney suffered more damage than those in WT kidneys. When interstitial immune cells were examined, the contact frequency of macrophages with infiltrated T cells increased and the immunometabolic features of T cells changed to showing high oxidative phosphorylation and fatty acid metabolism and overproduction of IFN-γ. The Vsir–/– parenchymal tissue cells responded to this altered milieu of interstitial immune cells as more IL-9 was produced, which augmented tubulointerstitial fibrosis. Blocking antibodies against IFN-γ and IL-9 protected the above pathological process in VISTA-depleted conditions. In human samples with acute glomerular injury (e.g., antineutrophil cytoplasmic autoantibody vasculitis), high VISTA expression in tubulointerstitial immune cells was associated with low tubulointerstitial fibrosis and good prognosis. Therefore, VISTA is a sentinel protein expressed in kidney macrophages that prevents tubulointerstitial fibrosis via the IFN-γ/IL-9 axis after acute antibody-mediated glomerular injury.