Immune Response to Persistent Staphyloccocus Aureus Periprosthetic Joint Infection in a Mouse Tibial Implant Model.

Staphyloccocus aureus is one of the major pathogens in orthopedic periprosthetic joint infection (PJI), a devastating complication of total joint arthroplasty that often results in chronic and persistent infections that are refractory to antibiotics and require surgical interventions. Biofilm formation has been extensively investigated as a reason for persistent infection. The cellular composition, activation status, cytokine profile, and role of the immune response during persistent S. aureus PJI are incompletely understood. In this study, we used histology, multiparametric flow cytometry, and gene expression analysis to characterize the immune response in a clinically relevant orthopedic PJI model. We tested the hypothesis that persistent S. aureus infection induces feedback mechanisms that suppress immune cell activation, thereby affecting the course of infection. Surprisingly, persistent infection was characterized by strikingly high cytokine gene expression indicative of robust activation of multiple components of innate and adaptive immunity, along with ongoing severe neutrophil-dominated inflammation, in infected joint and bone tissues. Activation and expansion of draining lymph nodes and a bone marrow stress granulopoiesis reaction were also maintained during late phase infection. In parallel, feedback mechanisms involving T-cell inhibitory receptors and exhaustion markers, suppressive cytokines, and regulatory T cells were activated and associated with decreased T-cell proliferation and tissue infiltration during the persistent phase of infection. These results identify the cellular and molecular components of the mouse immune response to persistent S. aureus PJI and indicate that neutrophil infiltration, inflammatory cytokine responses, and ongoing lymph node and bone marrow reactions are insufficient to clear infection and that immune effector mechanisms are suppressed by feedback inhibitory pathways. These immune-suppressive mechanisms are associated with diminished T-cell proliferation and tissue infiltration and can be targeted as part of adjuvant immunotherapeutic strategies in combination with debridement of biofilm, antibiotics, and other therapeutic modalities to promote eradication of infection. © 2021 American Society for Bone and Mineral Research (ASBMR).

Immune and repair responses in joint tissues and lymph nodes after knee arthroplasty surgery in mice.

The importance of a local tissue immune response in healing injured tissues such as skin and lung is well established. Little is known about whether sterile wounds elicit lymph node (LN) responses and inflammatory responses after injury of musculoskeletal tissues that are mechanically loaded during the repair response. We investigated LN and tissue immune responses in a tibial implant model of joint replacement surgery where wounded tissue is subjected to movement and mechanical loading postoperatively. Draining inguinal and iliac LNs expanded postoperatively, including increases in regulatory T cells and activation of a subset of T cells. Thus, tissue injury was actively sensed in secondary lymphoid organs, with the potential to activate adaptive immunity. Joint tissues exhibited three temporally distinct immune response components, including a novel interferon (IFN) response with activation of signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) pathways. Fibrovascular tissue formation was not associated with a macrophage type 2 (M2) reparative immune response, but instead with delayed induction of interleukin-1 family (IL-1ß, IL-33, IL-36), IL-17, and prostaglandin pathway genes concomitant with transforming growth factor (TGF)-ß and growth factor signaling, fibroblast activation, and tissue formation. Tissue remodeling was associated with activity of the HOX antisense intergenic RNA (HOTAIR) pathway. These results provide insights into immune responses and regulation of tissue healing after knee arthroplasty that potentially can be used to develop therapeutic strategies to improve healing, prevent arthrofibrosis, and improve surgical outcomes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Mortalin/glucose-regulated protein 75 promotes the cisplatin-resistance of gastric cancer via regulating anti-oxidation/apoptosis and metabolic reprogramming.

Platinum drug treatment is one of the most predominant chemotherapeutic strategies for patients with gastric cancer (GC). However, the therapeutic effect is less than satisfactory, largely due to the acquired resistance to platinum drugs. Therefore, a better understanding of the underlying mechanisms can greatly improve the therapeutic efficacy of GC. In this study, we aimed to investigate the chemo-resistance related functions/mechanisms and clinical significance of glucose-regulated protein 75 (GRP75) in GC. Here, our data showed that compared with SGC7901 cells, the expression of GRP75 was markedly higher in cisplatin-resistance cells (SGC7901CR). Knockdown of GRP75 abolished the maintenance of mitochondrial membrane potential (MMP) and inhibited the nuclear factor erythroid-2-related factor 2 (NRF2), phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT), hypoxia-inducible factor 1α (HIF-1α), and c-myc, which resulted in blocking the activation of their downstream targets. These processes attenuated the anti-oxidation/apoptosis abilities and altered the metabolic reprogramming in SGC7901CR cells, leading to re-sensitizing these cells to cisplatin. However, overexpression of GRP75 in SGC7901 cells caused the opposite effects. A xenografts model confirmed the abovementioned results. In GC patients receiving platinum chemotherapy and a meta-analysis, a high level of GRP75 was positively associated with aggressive characteristics and poor prognosis including but not limited to gastrointestinal cancers, and was an independent predictor for overall survival. Collectively, our study indicated that GRP75 was involved in the cisplatin-resistance of GC and that GRP75 could be a potential therapeutic target for restoring the drug response in platinum-resistance cells and a useful additive prognostic tool in guiding clinical management of GC patients.

Identification of a novel pathogenic MLH1 mutation and recommended genetic screening strategy: An investigation of three Chinese Lynch syndrome pedigrees.

BACKGROUND: Lynch syndrome (LS) is an autosomal-dominant disorder that increases the risk of many cancers. The genetic basis of LS is germline mutations in DNA mismatch repair genes. METHODS: We performed next-generation sequencing on blood cells obtained from the members of three unrelated LS pedigrees. Immunohistochemistry staining was performed to analyze protein expression. RESULTS: Multigene panel screening revealed three mutL homolog 1 (MLH1) pathogenic mutations (c.199G>A, c.790 + 1G>A, and c.1557_1558 + 8delGGGTACGTAA, unreported) confirmed by Sanger sequencing. Immunohistochemistry showed a loss of MLH1 protein expression. We also confirmed that the unreported mutant allele was inherited for at least three generations. CONCLUSION: These results provide new insights into the molecular mechanisms underlying the pathogenicity of MLH1 mutations and reaffirm the importance of genetic screening for the early diagnosis of LS.

Reversal of sorafenib resistance in hepatocellular carcinoma: epigenetically regulated disruption of 14-3-3η/hypoxia-inducible factor-1α.

Sorafenib resistance is one of the main obstacles to the treatment of advanced/recurrent hepatocellular carcinoma (HCC). Here, sorafenib-resistant HCC cells and xenografts in nude mice were used as experimental models. A cohort of patients with advanced recurrent HCC who were receiving sorafenib therapy was used to assess the clinical significance of this therapy. Our data showed that 14-3-3η maintained sorafenib resistance in HCC. An analysis of the underlying molecular mechanisms revealed that 14-3-3η stabilizes hypoxia-inducible factor 1α (HIF-1α) through the inhibition of ubiquitin-dependent proteasome protein degradation, which leads to the maintenance of cancer stem cell (CSC) properties. We further found that microRNA-16 (miR-16) is a competent miRNA that reverses sorafenib resistance by targeting the 3'-UTR of 14-3-3η and thereby inhibits 14-3-3η/HIF-1α/CSC properties. In HCC patients, significant negative correlations were found between the expression of miR-16 and 14-3-3η, HIF-1α, or CSC properties. Further analysis showed that low miR-16 expression but high 14-3-3η expression can prognosticate sorafenib resistance and poor survival. Collectively, our present study indicated that miR-16/14-3-3η is involved in sorafenib resistance in HCC and that these two factors could be potential therapeutic targets and biomarkers for predicting the response to sorafenib treatment.

Arsenic trioxide reverses the chemoresistance in hepatocellular carcinoma: a targeted intervention of 14-3-3η/NF-κB feedback loop.

BACKGROUND: Multi-drug resistance (MDR) is one of the main obstacles for treatment of advanced/recurrent hepatocellular carcinoma (HCC). We have previously identified arsenic trioxide (ATO) as an effective metastasis/angiogenesis inhibitor in HCC. Here, we further found that MDR-HCC cells were more sensitive to ATO. METHODS: The MDR-HCC cells were used as experimental models. Biological functions were investigated using cell transfection, polymerase chain reaction, western blot, southwestern blot, immunostaining, immunoprecipitation plus atomic fluorescence spectrometry, and so on. RESULTS: The MDR-HCC cells underwent high oxidative stress condition, and employed adaptive mechanisms for them to survive; while ATO abolished such mechanisms via targeting the 14-3-3η/nuclear factor kappa B (NF-κB) feedback Loop. Briefly, in MDR cells, the increase of ROS activated NF-κB signaling, which transcriptionally activated 14-3-3η. Meanwhile, the activation of NF-κB can be constitutively maintained by 14-3-3η. As a NF-κB inhibitor, ATO transcriptionally inhibited the 14-3-3η mRNA level. Meanwhile, ATO was also validated to directly bind to 14-3-3η, enhancing the degradation of 14-3-3η protein in an ubiquitination-dependent manner. Knockdown of 14-3-3η reduced the ATO-induced reversal extents of drug resistance in MDR cells. CONCLUSION: 14-3-3η/NF-κB feedback loop plays an important role in maintaining the MDR phenotype in HCC. Moreover, via targeting such feedback loop, ATO could be considered as a potential molecular targeted agent for the treatment of HCC.

Diet-induced obesity alters the maternal metabolome and early placenta transcriptome and decreases placenta vascularity in the mouse.

Maternal obesity is associated with an increased risk of obesity and metabolic disease in offspring. Increasing evidence suggests that the placenta plays an active role in fetal programming. In this study, we used a mouse model of diet-induced obesity to demonstrate that the abnormal metabolic milieu of maternal obesity sets the stage very early in pregnancy by altering the transcriptome of placenta progenitor cells in the preimplantation (trophectoderm [TE]) and early postimplantation (ectoplacental cone [EPC]) placenta precursors, which is associated with later changes in placenta development and function. Sphingolipid metabolism was markedly altered in the plasma of obese dams very early in pregnancy as was expression of genes related to sphingolipid processing in the early placenta. Upregulation of these pathways inhibits angiogenesis and causes endothelial dysfunction. The expression of many other genes related to angiogenesis and vascular development were disrupted in the TE and EPC. Other key changes in the maternal metabolome in obese dams that are likely to influence placenta and fetal development include a marked decrease in myo and chiro-inositol. These early metabolic and gene expression changes may contribute to phenotypic changes in the placenta, as we found that exposure to a high-fat diet decreased placenta microvessel density at both mid and late gestation. This is the first study to demonstrate that maternal obesity alters the transcriptome at the earliest stages of murine placenta development.

Adverse metabolic phenotype of female offspring exposed to preeclampsia in utero: a characterization of the BPH/5 mouse in postnatal life.

Preeclampsia (PE) is a devastating disorder of pregnancy that classically presents with maternal hypertension and proteinuria after 20 wk of gestation. In addition to being a leading cause of maternal and fetal morbidity/mortality, epidemiological and prospective studies have revealed long-term consequences for both the mother and baby of preeclamptic pregnancies, including chronic hypertension as well as other cardiovascular diseases and metabolic derangements. To better understand the effect of in utero exposure of PE on offspring, we utilized the BPH/5 mouse, a spontaneous model of the maternal and fetal PE syndrome. We hypothesized that young BPH/5 offspring would have altered metabolic and cardiovascular phenotypes. Indeed, BPH/5 growth-restricted offspring showed excess catch-up growth by early adulthood due to hyperphagia and increased white adipose tissue (WAT) accumulation, with inflammation markers isolated to the reproductive WAT depot only. Both excessive WAT accumulation and the inflammatory WAT phenotype were corrected by pair-feeding young BPH/5 female mice. We also found that young BPH/5 female mice showed evidence of leptin resistance. Indeed, chronic hyperleptinemia has been shown to characterize other rodent models of PE; however, the maternal metabolic profile before pregnancy has not been fully understood. Furthermore, we found that these mice show signs of cardiovascular anomalies (hypertension and cardiomegaly) and altered signaling within the reproductive axis in early life. Future studies will involve challenging the physiological metabolic state of BPH/5 mice through pair-feeding to reduce WAT before pregnancy and determining its causal role in adverse pregnancy outcomes.