Expression Of Allograft Inflammatory Factor-1 Research Articles

Identification of allograft inflammatory factor-1 suppressing the progression and indicating good prognosis of osteosarcoma

BackgroundOsteosarcoma is one of the most common cancers worldwide. Intense efforts have been made to elucidate the pathogeny, but the mechanisms of osteosarcoma are still not well understood. We aimed to investigate the potential biomarker, allograft inflammatory factor-1 (AIF1), affecting the progression and prognosis of osteosarcoma.MethodsThree microarray datasets were downloaded from GEO datasets and one was obtained from the TCGA dataset. The differentially expressed genes (DEGs) were identified. GO and KEGG functional enrichment analyses of overlapped DEGs were performed. The PPI network of overlapped DEGs was constructed by STRING and visualized with Cytoscape. Overall survival (OS) and Metastasis free survival (MFS) were analyzed from GSE21257. Finally, the effect of the most relevant core gene affecting the progression of osteosarcoma was examined in vitro.ResultsOne hundred twenty six DEGs were identified, consisting of 65 upregulated and 61 downregulated genes. Only AIF1 was significantly associated with OS and MFS. It was found that AIF1 could be enriched into the NF-κB signaling pathway. GSEA and ssGSEA analyses showed that AIF1 was associated with the immune invasion of tumors. Cell experiments showed that AIF1 was underexpressed in osteosarcoma cell lines, while the malignant propriety was attenuated after overexpressing the expression of AIF1. Moreover, AIF1 also affects the expression of the NF-κB pathway.ConclusionIn conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the carcinogenesis and progression of osteosarcoma, and provide candidate targets for diagnosis and treatment of osteosarcoma.

Histopathological Clues of Enhanced Inflammation in the Placental Tissue of Women with Chronic Venous Disease in Lower Limbs during Pregnancy.

It is estimated that approximately one in three women develop chronic venous disease (CVD) during pregnancy, a broad spectrum of morphofunctional disorders affecting the venous system in different regions of the body, including the lower limbs. A growing body of evidence supports the diverse maternofetal consequences derived from this condition, with the placenta being an organ particularly affected. Among other consequences, having CVD during pregnancy has been associated with systemic inflammation and altered cytokines and chemokine profiles in the maternal and fetal serum related to this condition. In the present work, we aimed to analyze if these inflammatory changes also occurred in the placental tissue of women with CVD, exploring by immunohistochemistry and real-time PCR (RT-qPCR) gene and protein expression of critical inflammatory markers like allograft inflammatory factor 1 (AIF-1), interleukin 10 (IL-10), IL-12A, and IL-18. Our results demonstrate an enhanced tissue expression of AIF-1, IL-12A, and IL-18, accompanied by a decrease in IL-10 in the placentas of women who had undergone CVD during pregnancy. Overall, our results suggest a possible pathophysiological role of inflammation in the placental tissue of women with CVD during pregnancy, although the precise consequences of this feature remain to be deeply analyzed.

CKLF induces microglial activation via triggering defective mitophagy and mitochondrial dysfunction

ABSTRACT Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases. Abbreviation: 3-MA: 3-methyladenine; AIF1: allograft inflammatory factor 1; ANOVA: analysis of variance; BAF: bafilomycin A1; BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CKLF/CKLF1: chemokine-like factor 1; CNS: central nervous system; DMEM: Dulbecco’s Modified Eagle Medium; DNM1L: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; IRF3: interferon regulatory factor 3; IgG: immunoglobulin G; LAMP1: lysosomal-associated membrane protein 1; LAPTM4A: lysosomal-associated protein transmembrane 4A; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; mRFP: monomeric red fluorescent protein; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; OPTN: optineurin; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PINK1: PTEN induced putative kinase 1; PLL: poly-L-lysine; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; VDAC: voltage-dependent anion channel

Chronic Venous Disease during Pregnancy Is Related to Inflammation of the Umbilical Cord: Role of Allograft Inflammatory Factor 1 (AIF-1) and Interleukins 10 (IL-10), IL-12 and IL-18.

Chronic venous disease (CVD) is a common condition that affects the veins in the lower limbs, resulting in a variety of symptoms, such as swelling, pain, and varicose veins (VVs). The plenty hormonal, hemodynamic and mechanical changes occurred in pregnancy make women especially vulnerable to suffer from this condition in this period. Previous works have identified that CVD is associated with an increased inflammatory milieu and significant damage in maternofetal tissues, such as the umbilical cord. However, the inflammatory status of this structure in these patients has not been studied yet. Thus, the aim of the present study was to examine gene and protein expression of a set of inflammatory markers-Allograft inflammatory factor 1 (AIF-1), the proinflammatory cytokines interleukin 12A (IL-12A) and IL-18 and the anti-inflammatory product IL-10-in the umbilical cord of women with CVD during pregnancy (N = 62) and healthy pregnant women (HC; N = 52) by the use of real time qPCR and immunohistochemistry (IHC). Our results demonstrate that the umbilical cord tissue from CVD women exhibit an increased expression of AIF-1, IL-12A and IL-18 along with a decrease in IL-10. Therefore, our study suggests an inflammatory status of this structure related to CVD. Further studies should be conducted to evaluate the expression of other inflammatory markers, as well as to analyze the maternofetal impact of these findings.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

Allograft inflammatory factor 1 is a potential diagnostic, immunological, and prognostic biomarker in pan-cancer.

Allograft Inflammatory Factor 1 (AIF-1) is a member of the allograft inflammatory factor gene family and plays an essential role in the occurrence and development of malignant tumors. However, little is known about the expression pattern, predictive value, and biological function of AIF-1 across cancers. We first analyzed AIF-1 expression across cancers based on data from public databases. Univariate Cox regression and Kaplan-Meier analyses were used to explore the predictive value of AIF-1 expression in various cancers. Moreover, gene set enrichment analysis (GSEA) was applied to determine the cancer hallmarks associated with AIF-1 expression. Spearman correlation analysis was performed to investigate the association between AIF-1 expression and tumor microenvironment scores, immune cell infiltration, immune-related genes, TMB, MSI, and DNA methyltransferases. AIF-1 expression was upregulated in most cancer types and exhibited prognosis-predictive ability. AIF-1 expression was positively correlated with immune infiltrating cells and immune checkpoint-related genes in most cancers. Additionally, the promoter methylation level of AIF-1 was different in distinct tumors. High methylation levels of AIF-1 were associated with a worse prognosis in UCEC and melanoma, whereas they were associated with a better prognosis in GBM, KIRC, OV, and UVM. Finally, we found that AIF-1 was significantly highly expressed in KIRC tissues. Functionally, silencing AIF-1 dramatically decreased proliferation, migration, and invasion abilities. Our results reveal that AIF-1 acts as a robust tumor biomarker and is closely correlated with tumor immune infiltration. Furthermore, AIF-1 may function as an oncogene and promote tumor progression in KIRC.

Allograft inflammatory factor-1 released from the cerebral microglia affect several organs in the body

Allograft inflammatory factor-1 (AIF-1) is expressed in microglia. Unilateral common carotid artery occlusion (UCCAO) was conducted to elucidate mechanisms that regulate AIF-1 expression in C57BL/6 male mice. Immunohistochemical reactivity of microglia against anti-AIF-1 antibody was increased significantly in the brain of this model. The increased AIF-1 production was further confirmed by ELISA using brain homogenate. Real-time PCR demonstrated that the increased AIF-1 production was regulated at the transcriptional level. Serum AIF-1 levels were further examined by ELISA and marked increase was observed on Day 1 of UCCAO. To examine the influence of AIF-1, immunohistochemical staining was performed and revealed that the immunoreactivity against anti-Iba-1 antibody was significantly increased in various organs. Among them, the accumulation of Iba-1+ cells were observed prominently in the spleen. Intraperitoneal injection of minocycline, a potent microglia inhibitor, reduced the number of Iba-1+ cells suggesting microglia activation-dependent accumulation. Based on these results, AIF-1 expression was further examined in the murine microglia cell line MG6. AIF-1 mRNA expression and secretion were up-regulated when the cells were cultured under hypoxic condition. Importantly, stimulation of the cells with recombinant AIF-1 induced the expression of AIF-1 mRNA. These results may suggest that increased AIF-1 production by microglia in cerebral ischemia regulate the AIF-1 mRNA expression at least in part by an autocrine manner.

Allograft inflammatory factor-1 stimulates inflammatory properties of peripheral blood leukocytes and increases cell viability via enhancing mitochondrial function in Ctenopharyngodon idellus

Allograft inflammatory factor-1 (AIF-1) is a 17 kDa calcium-binding protein associated with numerous inflammatory diseases. The full-length cDNA of AIF-1 has been identified in grass carp, Ctenopharyngodon idellus in our previous study, and it was assumed to be a novel molecule involved in immune responses. To clarify this aspect, the level of AIF-1 expression was amplified and reduced in grass carp peripheral blood leukocytes via transfection of vector pcDNA3.1-AIF1-EGFP and pLKO.1-shRNA-EGFP-puro, respectively. Thereafter, AIF-1 stimulated cell proliferation, inhibited cell apoptosis, which might benefit from improved mitochondrial function as evidenced by increased mitochondrial membrane potential, subsequently promoted ATP production. In addition, AIF-1 induced leukocyte migration via up-regulated monocyte chemotactic protein-1(MCP-1) secretion, enhanced neutral red uptake into leukocyte, provoked pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL1β), interleukin 6 (IL6), interleukin 8 (IL8) and suppressed anti-inflammatory cytokine interleukin 10 (IL10) production. These results indicated AIF-1 played a critical role in grass carp innate immune system.

Allograft inflammatory factor-1 enhances inflammation and oxidative stress via the NF-κB pathway in diabetic kidney disease

Inflammation and glomerular endothelial dysfunction promote diabetic kidney disease (DKD) progression, but the mechanisms are not fully understood. Allograft inflammatory factor-1 (AIF-1) is a protein that regulates inflammatory reactions and immune responses. This study aimed to explore the mechanism of AIF-1 in a DKD animal model and mouse renal glomerular endothelial cells (MRGECs). We injected AIF-1-shRNA into the tail vein to knockdown AIF-1 in db/db mice. Metabolic index, renal pathological changes and inflammatory factors were measured in each group. Lentiviral transfection was used to overexpress AIF-1 in MRGECs. Inflammatory factors, oxidative stress and nuclear factor-κB (NF-κB) pathway-related proteins were examined. AIF-1 expression was upregulated in glomerular endothelial cells in renal tissues of db/db mice. Knockdown of AIF-1 reversed kidney injury and renal inflammation in db/db mice. In a 30 mM high-glucose environment, overexpression of AIF-1 in MRGECs activated the NF-κB pathway and induced inflammation and oxidative stress. Moreover, this damage could be attenuated by the addition of an NF-κB inhibitor (BAY 11–7082). In conclusion, AIF-1 facilitates glomerular endothelial cell inflammation and oxidative stress in DKD via the NF-κB signaling pathway. Our results provide evidence for the molecular mechanism of DKD and may offer a potential target for DKD treatment.

Associations of Biomarkers of Inflammation and Breast Cancer in the Breast Adipose Tissue of Women with Combined Measures of Adiposity.

Background Mechanisms underlying the obesity-breast cancer link involve inflammation but need to be elucidated. Determining obesity by combining body mass index (BMI) with the waist circumference (WC) may clarify the role of inflammatory and hormonally related markers in breast cancer. We examined the effect of combining adiposity indices (BMI/WC) with the gene expression of several biomarkers involved in breast cancer. Methods Expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), estrogen receptor-alpha (ER-α), allograft inflammatory factor 1 (AIF1), cyclooxygenase-2 (COX2), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and leptin (LEP) in 141 adipose breast tissues was quantified using qPCR method. BMI and WC were measured by a trained nurse and categorized using the median split, BMILOWCLO, BMILOWCHI, BMIHIWCLO, and BMIHIWCHI. Results Gene expression of IL-6 (3-fold), TNF-α (2-fold), and LEP (2-fold) was higher in the breast adipose tissue of women with high WC regardless of BMI, that is, BMILOWCHI and BMIHIWCHI women (all P < 0.01). Compared to BMILOWCLO women, gene expression of CYP19A1, COX2, and AIF1 was increased by two-fold in breast adipose tissue of BMIHIWCHI women (P < 0.10). ER-α was not different across adiposity categories. Conclusions The expression of some biomarkers, particularly those related to inflammation, is elevated in breast adipose tissue of women with a high WC independent of BMI. Obesity monitoring should also include women with normal or low BMI, but with central adiposity.

Quantification of the inflammatory responses to pro-and anti-inflammatory agents in Manila clam, Ruditapes philippinarum

Inflammation is a form of innate immune response of living organisms to harmful stimuli. In marine bivalves, inflammation is a common defense mechanism. Several studies have investigated the morphological features of inflammation in bivalves, such as hemocyte infiltration. However, the molecular and biochemical responses associated with inflammation in marine bivalves remain unexplored. Here, we investigated changes in nitric oxide (NO) levels, cyclooxygenase 2 (COX-2) activity, and allograft inflammatory factor-1 (AIF-1) gene expression levels in hemolymph samples collected from Manila clam (Ruditapes philippinarum) exposed to pro- and anti-inflammatory substances. These included the pro-inflammatory agent lipopolysaccharide (LPS), and the nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac, all widely used in vertebrates. Our study showed that NO levels, COX-2 activity, and AIF-1 expression increased in response to the treatments with LPS and decreased in response to the treatments with NSAIDs in a concentration-dependent manner. These results suggest that the mechanism of inflammatory responses in bivalves is very similar to that of vertebrates, and we propose that inflammatory responses can be quantified using these techniques and used to determine the physiological status of marine bivalves exposed to biotic or abiotic stresses.

Leishmania donovani infection suppresses Allograft Inflammatory Factor-1 in monocytes and macrophages to inhibit inflammatory responses

Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.

IMMU-29. AIF1 IS A PROGNOSTIC BIOMARKER AND CORRELATED WITH IMMUNE INFILTRATES IN GLIOMAS

Gliomas remain highly variable clinical behaviors, leading to emerging studies to identify prognostic factors. AIF1 (Allograft Inflammatory Factor 1) is critical for promoting both macrophage- and dendritic cells (DCs)-mediated inflammatory response and growth of vascular smooth muscle cells and T-lymphocytes. Through comparative analyses of primary LGG patients from The Cancer Genome Atlas (TCGA) dataset and Chinese Glioma Genome Atlas(CGGA) dataset, we reported that the expression level and methylation level of AIF1 gene vary among glioma patients and AIF1 expression or gene body methylation is significantly associated with glioma patient survival. Cox regression results confirmed that AIF1 played an independent predictor of survival in lower-grade glioma(LGG), with a cox coefficient of 0.251 indicating a worse prognosis. Moreover, AIF1 expression was positively correlated with infiltrating levels of CD4+ T and B cells, macrophages, neutrophils, and DCs in LGG and glioblastoma(GBM). AIF1 expression also showed strong correlations with specific immune cell markers in LGG and GBM. In addition, AIF1 expression potentially contributed to the regulation of glioma-associated macrophages and microglia. In conclusion, our findings suggested that AIF1 was correlated with prognosis and immune infiltrating levels, and it can be used as a prognostic factor in gliomas.

Allograft Inflammatory Factor-1 in Metazoans: Focus on Invertebrates.

Simple SummaryDuring their life, all living organisms defend themselves from pathogens using complex strategies. Vertebrates and invertebrates share mechanisms and molecules that guarantee their overall bodily integrity. Allograft inflammatory factor-1 (AIF-1) is a protein extensively studied in vertebrates, and especially in mammals. This factor, generally involved in inflammation events occurring upon pathogenic infection or tissue injury, is linked to several important human diseases. This review collects data on the presence and role of AIF-1 in invertebrates, which are still poorly investigated organisms. Multiple alignment and phylogenetic analysis reveal that AIF-1 is conserved in vertebrates and invertebrates, suggesting similarity of functions. In some invertebrate species, the expression of AIF-1 increases considerably after a bacterial challenge, indicating that it plays a key role during the immune responses. This review highlights the importance of studying this protein in invertebrates as a way to improve our knowledge of innate immunity mechanisms and to better understand inflammatory regulation events in mammals.Allograft inflammatory factor-1 (AIF-1) is a calcium-binding scaffold/adaptor protein often associated with inflammatory diseases. Originally cloned from active macrophages in humans and rats, this gene has also been identified in other vertebrates and in several invertebrate species. Among metazoans, AIF-1 protein sequences remain relatively highly conserved. Generally, the highest expression levels of AIF-1 are observed in immunocytes, suggesting that it plays a key role in immunity. In mammals, the expression of AIF-1 has been reported in different cell types such as activated macrophages, microglial cells, and dendritic cells. Its main immunomodulatory role during the inflammatory response has been highlighted. Among invertebrates, AIF-1 is involved in innate immunity, being in many cases upregulated in response to biotic and physical challenges. AIF-1 transcripts result ubiquitously expressed in all examined tissues from invertebrates, suggesting its participation in a variety of biological processes, but its role remains largely unknown. This review aims to present current knowledge on the role and modulation of AIF-1 and to highlight its function along the evolutionary scale.

Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes.

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Role of allograft inflammatory factor-1 in the regulation of inflammation and oxidative stress in primary peritoneal mesothelial cells.

Peritoneal dialysis (PD) is often used to treat patients with end stage renal disease, and its long-term complications include excessive inflammation and oxidative stress. Allograft inflammatory factor 1 (AIF-1), as a cytoplasmic protein, is originally identified from infiltrating macrophages, and it was associated with inflammation in the cells other than macrophages, such as endothelial cells and vascular smooth muscle cells. To clarify the molecular mechanisms of AIF-1-modulated pathological changes in the peritoneum during PD, we first detected the AIF-1 expression in peritoneal tissues from PD mice. Results revealed that the pro-fibrotic stimulation caused AIF-1 upregulation and triggered inflammation in peritoneal tissues, and that AIF-1 co-expressed with pan-cytokeratin (a marker of peritoneal mesothelial cells). We next treated primary mouse peritoneal mesothelial cells (pan-cytokeratin and intercellular adhesion molecule 1 positive cells) with 50 or 100 ng/mL recombinant AIF-1, and evaluated the direct effects of AIF-1 on these cells in vitro. We found that exogenous AIF-1 treatment induced inflammation and oxidative stress in mesothelial cells. Apart from the augmented IL-6 and TNF-α secretion, the level of ROS was upregulated and the activity of anti-oxidative SOD was reduced in cells exposed to AIF-1. Moreover, AIF-1 simulation triggered the activation of NF-κB pathway-enhanced the conversion of IκB to phosphorylated IκB and promoted the translocation of NF-κB p65 from cytoplasm into nucleus. Additionally, AIF-1-evoked inflammation in peritoneal mesothelial cells was attenuated by the addition of NF-κB inhibitor (BAY 11-7082). In brief, this study provides us novel information to understand the molecular regulation mechanisms of AIF-1 in peritoneal fibrosis.

Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro.

The multistep differentiation process from hematopoietic stem cells through common myeloid progenitors into committed dendritic cell (DC) subsets remains to be fully addressed. These studies now show that Allograft Inflammatory Factor-1 (AIF1) is required for differentiation of classical DC type 1 (cDC1) subsets and monocyte-derived DC (Mo-DC). Phenotypic studies found that AIF1 expression increased in committed subsets differentiating from common myeloid progenitors (CMP). However, silencing AIF1 expression in hematopoietic stem progenitors restrained the capacity to differentiate into Mo-DC and cDC1 cell subsets under GM-CSF or Flt3-L stimuli conditions, respectively. This was further marked by restrained expression of IRF8, which is critical for development of Mo-DC and cDC1 subsets. As a result, absence of AIF1 restrained the cells at the Lin−CD117+FcγR−CD34+ CMP stage. Further biochemical studies revealed that abrogating AIF1 resulted in inhibition of the NFκB family member RelB expression and p38 MAPK phosphorylation during differentiation of Mo-DC. Lastly, protein binding studies identified that AIF1 interacts with protein kinase C (PKC) to influence downstream signaling pathways. Taken together, this is the first report showing a novel role of AIF1 as a calcium-responsive scaffold protein that supports IRF8 expression and interacts with PKC to drive NFκB-related RelB for successfully differentiating hematopoietic progenitor cells into cDC and Mo-DC subsets under Flt3-L and GM-CSF stimuli, respectively.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1.

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

The Role of Allograft Inflammatory Factor-1 in the Effects of Experimental Diabetes on B Cell Functions in the Heart.

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. However, the etiology of the cardiac disease is unknown. Therefore, we assessed the gene expression in the left ventricle of diabetic and non-diabetic mice using Affymetrix microarray analysis. Allograft inflammatory factor-1 (AIF-1), one of the top downregulated B cell inflammatory genes, is associated with B cell functions in inflammatory responses. Real-time reverse transcriptase-polymerase chain reaction confirmed the Affymetrix data. The expression of CD19 and AIF-1 were downregulated in diabetic hearts as compared to control hearts. Using in vitro migration assay, we showed for the first time that AIF-1 is responsible for B cell migration as B cells migrated to GFP-AIF-1-transfected H9C2 cells compared to empty vector-transfected cells. Interestingly, overexpression of AIF-1 in diabetic mice prevented streptozotocin-induced cardiac dysfunction, inflammation and promoted B cell homing into the heart. Our results suggest that AIF-1 downregulation inhibited B cell homing into diabetic hearts, thus promoting inflammation that leads to the development of diabetic cardiomyopathy, and that overexpression of AIF-1 could be a novel treatment for this condition.

Expression of Allograft Inflammatory Factor-1 (AIF-1) in Hepatocellular Carcinoma.

BackgroundAllograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein cloned from activated macrophages in human and rat allografts. AIF-1 has been identified as a modulator of inflammatory response, and recently published studies have shown its increased expression in carcinogenesis. However, there are still limited data on the potential functional role of AIF-1 in hepatocellular carcinoma (HCC).Material/MethodsWe evaluated the expression of AIF-1 in 104 cases of paired HCC and adjacent non-cancerous liver tissues using immunohistochemistry, Western blotting, and qPCR analysis, and sought to determine whether its expression was correlated with clinicopathological features. In vitro assays, including cell proliferation and migration assays, were used to study the effects of AIF-1 knockdown in L02 human hepatocyte, and Huh7 and SMMC7721 liver cancer cell lines.ResultsExpression of AIF-1 was increased in HCC compared to adjacent normal liver tissues and was positively correlated with median tumor size (p=0.046), number of tumor deposits (p=0.009), the Barcelona Clinic Liver Cancer (BCLC) stage (p=0.004), and portal vein tumor thrombus (PVTT) (p<0.001). Huh7 and SMMC7721 human HCC cells demonstrated upregulated AIF-1 expression compared to normal hepatocytes. Small interfering RNA (siRNA)-mediated silencing of AIF-1 expression resulted in a reduction in cell proliferation and migration in human HCC cells.ConclusionsThese findings suggest AIF-1 may have roles as a diagnostic or prognostic biomarker and a promising therapeutic target in HCC.