Age-related chronic low-grade inflammation contributes to both frailty and bone loss. One of the key regulators of inflammatory signaling that declines with age is tristetraprolin (TTP), an RNA-binding protein that promotes degradation of pro-inflammatory transcripts. In this study, we investigated whether stabilizing TTP during aging could reduce frailty and enhance bone health by mitigating inflammation and immune dysfunction. We utilized a knock-in mouse model (TTP triangle ARE), in which an AU-rich region of the 3 ' untranslated region was deleted to stabilize TTP mRNA and increase protein expression. Aged TTP triangle ARE mice had reduced physical frailty scores, a composite measure based on body weight and physical performance, than age-matched wild-type controls (WT). Since frailty is associated with fracture risk, we examined bone structure. Aged TTP triangle ARE males exhibited significantly higher bone mineral density and improved bone microarchitecture relative to WT mice. Our prior work showed that aging elevates myeloid-derived suppressor cells (MDSCs), which possess osteoclastogenic potential. The monocytic MDSCs (M-MDSCs) from the bone marrow of aged TTP triangle ARE formed fewer osteoclasts than those from WT mice. Further, transcriptomic analysis of M-MDSCs revealed downregulation of bone resorption and remodeling pathways, along with upregulation of immune activation genes. In addition, immunophenotyping revealed a healthier, youthful-like immune profile in aged TTP triangle ARE mice, including increased T-cell reservoirs. These findings signify the critical role of TTP in bone health during aging by regulating osteoimmunological induction of M-MDSCs, which leads to a partial reversal of the age-associated immune senescent phenotype, resulting in increased bone mineral density and improved functional capacity during aging.

前言:口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)是头颈部恶性肿瘤中最常见的病理类型。尽管在OSCC的诊断、手术和肿瘤治疗方面取得了进展,但过去30年中患者的5年生存率仍仅为50%左右。由于慢性炎症通常与较差的预后相关,因此了解肿瘤微环境在OSCC进展过程中的参与方式至关重要。肿瘤的内部结构比正常组织复杂得多,其特定的肿瘤微环境(tumor microenvironment,TME)由各种基质细胞和免疫细胞组成,并且贯穿从肿瘤早期发生到进展和转移的各个阶段。有研究报道,肿瘤相关巨噬细胞(tumor associated macrophages,TAMs)在与TME相关的免疫细胞中占恶性实体瘤体积的5-50%,参与调节癌症相关的炎症、免疫逃逸、基质重塑和癌症转移。来自TAMs的细胞因子和促炎因子在恶性转化的不同阶段发挥着关键作用。TAMs可分为两类,一类是经典激活的M1巨噬细胞,抑制肿瘤的发展;另一类是选择性激活的M2巨噬细胞,能够促进肿瘤发展,但是具体机制尚不清楚。双特异性磷酸酶-1(dual-specificity phosphatase-1,Dusp1),又称丝裂原活化蛋白激酶磷酸酶-1(mitogen-activated protein kinase phosphatase,Mkp-1),是最早发现的Mkps成员,其编码基因位于5q35.1,在人类细胞对环境应激反应以及细胞增殖的负调控中发挥重要作用。Dusp1通过去磷酸化丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPKs)上的苏氨酸-谷氨酸-酪氨酸基序,可以抑制p38、Erk和Jnk的表达。Dusp1已被证明参与不同的生物学过程,如代谢信号、骨骼肌功能、炎症反应和癌症发展。DUSP1基因在膀胱癌、乳腺癌、前列腺癌和结肠癌等多种癌症的原发灶中表达降低,同时在近80%的转移组织中也出现低表达。另有关于卵巢癌的研究表明,DUSP1可能在抑制丝足形成从而在细胞运动中起关键作用。虽然有研究报道在乳腺癌中,激活p38和Erk信号通路可诱导M2型巨噬细胞产生IL-8,促进肿瘤的迁移和侵袭。然而,目前对DUSP1在肿瘤组织中的研究大多局限于应用原位杂交和免疫组化检测其mRNA水平和蛋白表达水平,尚不清楚DUSP1在TME中是否直接作用于炎症免疫细胞发挥作用。前期我们课题组通过免疫组化、Western Blot和Realtime PCR检测了DUSP1在人OSCC组织中的表达情况,结果发现DUSP1在OSCC组织中呈低表达,且与肿瘤进展密切相关。结合前期研究结果及文献,本研究拟利用Dusp1全身敲除和骨髓条件性敲除小鼠模型研究肿瘤微环境中Dusp1缺失在口腔鳞状细胞癌的发生发展中的作用,并初步揭示DUSP1引导TAMs募集、极性和影响OSCC转移的机制,为OSCC靶向药物的研发奠定理论基础。材料与方法:1、实验材料:Dusp1基因全身敲除型小鼠(C57BL/6),Dusp1基因骨髓条件性敲除型小鼠(C57BL/6),小鼠同源口腔癌细胞系MOC2。2、实验方法:肿瘤细胞培养、TCGA数据库分析、小鼠背部皮下成瘤、小鼠口腔原位成瘤、4NQO药物诱导小鼠成瘤、免疫细胞分选、巨噬细胞培养、划痕实验、流式细胞仪技术、H&E染色、免疫组织化学染色、mRNA提取和Nanostring等。结果:1 DUSP1在TCGA数据库OSCC组织中表达水平分析结果在TCGA数据库中,比较DUSP1基因在口腔鳞癌和正常口腔黏膜组织中的表达情况。结果显示:与正常口腔黏膜组织相比,DUSP1在OSCC组织中表达呈显著性降低(P0.05)。接下来,我们设计实验制备Dusp1敲除小鼠和野生型小鼠肿瘤细胞单细胞悬液,使用CD45+显微磁珠标记后进行分选,发现在Dusp1敲除小鼠的肿瘤组织中,分选得到的CD45+炎性细胞较野生型显著增多(P0.05)。7.Dusp1通过TAMs对MOC2细胞迁移影响的检测结果通过对分选出的Dusp1敲除和野生型TAMs分别进行培养,而后提取无血清培养液离心后的上清液,使用该上清液进行MOC2细胞划痕实验。结果显示:与野生型小鼠TAMs上清液相比,Dusp1全身敲除小鼠TAMs上清液培养的MOC2细胞其划痕愈合速度显著加快(P<0.05)。8.Dusp1敲除小鼠TAMs差异表达基因的筛选与功能分析结果通过磁珠标记分选出TAMs进行mRNA提取和Nanostring实验,获得Dusp1敲除小鼠和野生型小鼠TAMs间显著差异的基因63个,其中高表达30个、低表达33个;根据GO分析等结果,选择若干差异显著的调控基因簇亚群进行再分析,发现这些基因在MAPK信号、Notch信号、骨髓来源相关基因和细胞因子/趋化因子信号、血管生成、代谢应急等相关信号通路中发挥重要作用。结论:1、Dusp1敲除促进口腔鳞状细胞癌的发生发展;2、Dusp1敲除促进肿瘤相关巨噬细胞向M2型转化,可能是OSCC转移的原因之一;3、Dusp1敲除可能通过影响多种骨髓来源基因、细胞因子、趋化因子等信号通路参与OSCC发生发展。

Oral squamous cell carcinomas (OSCC) constitute over 95% of all head and neck malignancies. As a key component of the tumor microenvironment (TME), chronic inflammation contributes towards the development, progression, and regional metastasis of OSCC. Tumor associated macrophages (TAMs) associated with OSSC promote tumorigenesis through the production of cytokines and pro-inflammatory factors that are critical role in the various steps of malignant transformation, including tumor growth, survival, invasion, angiogenesis, and metastasis. The mitogen-activated protein kinases (MAPKs) can regulate inflammation along with a wide range of cellular processes including cell metabolism, proliferation, motility, apoptosis, survival, differentiation and play a crucial role in cell growth and survival in physiological and pathological processes including innate and adaptive immune responses. Dual specificity MAPK phosphatases (MKPs) deactivates MAPKs. MKPs are considered as an important feedback control mechanism that limits MAPK signaling and subsequent target gene expression. This review outlines the role of MKP-1, the founding member of the MKP family, in OSCC and the TME. Herein, we summarize recent progress in understanding the regulation of p38 MAPK/MKP-1 signaling pathways via TAM-related immune responses in OSCC development, progression and treatment outcomes.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, invitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype. © 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

Intestinal colonization of the oral bacterium Haemophilus parainfluenzae has been associated with Crohn's disease (CD) severity and progression. This study examines the role of periodontal disease (PD) as a modifier for colonization of H. parainfluenzae in patients with CD and explores the mechanisms behind H. parainfluen-zae-mediated intestinal inflammation. Fifty subjects with and without CD were evaluated for the presence of PD, and their oral and fecal microbiomes were characterized. PD is associated with increased levels of H. parainfluenzae strains in subjects with CD. Oral inoculation of H. parainfluenzae elicits strain-dependent in-testinal inflammation in murine models of inflammatory bowel disease, which is associated with increased intestinal interferon-g (IFN-g)+ CD4+ T cells and disruption of the host hypusination pathway. In summary, this study establishes a strain-specific pathogenic role of H. parainfluenzae in intestinal inflammation and highlights the potential effect of PD on intestinal colonization by pathogenic H. parainfluenzae strains in pa-tients with CD.

Aging results in enhanced myelopoiesis, which is associated with an increased prevalence of myeloid leukemias and the production of myeloid-derived suppressor cells (MDSCs). Tristetraprolin (TTP) is an RNA binding protein that regulates immune-related cytokines and chemokines by destabilizing target mRNAs. As TTP expression is known to decrease with age in myeloid cells, we used TTP-deficient (TTPKO) mice to model aged mice to study TTP regulation in age-related myelopoiesis. Both TTPKO and myeloid-specific TTPKO (cTTPKO) mice had significant increases in both MDSC subpopulations M-MDSCs (CD11b(+)Ly6C(hi)Ly6G(-)) and PMN-MDSCs (CD11b(+)Ly6C(lo)Ly6G(+)), as well as macrophages (CD11b(+)F4/80(+)) in the spleen and mesenteric lymph nodes; however, no quantitative changes in MDSCs were observed in the bone marrow. In contrast, gain-of-function TTP knock-in (TTPKI) mice had no change in MDSCs compared with control mice. Within the bone marrow, total granulocyte-monocyte progenitors (GMPs) and monocyte progenitors (MPs), direct antecedents of M-MDSCs, were significantly increased in both cTTPKO and TTPKO mice, but granulocyte progenitors (GPs) were significantly increased only in TTPKO mice. Transcriptomic analysis of the bone marrow myeloid cell populations revealed that the expression of CC chemokine receptor 2 (CCR2), which plays a key role in monocyte mobilization to inflammatory sites, was dramatically increased in both cTTPKO and TTPKO mice. Concurrently, the concentration of CC chemokine ligand 2 (CCL2), a major ligand of CCR2, was high in the serum of cTTPKO and TTPKO mice, suggesting that TTP impacts the mobilization of M-MDSCs from the bone marrow to inflammatory sites during aging via regulation of the CCR2-CCL2 axis. Collectively, these studies demonstrate a previously unrecognized role for TTP in regulating age-associated myelopoiesis through the expansion of specific myeloid progenitors and M-MDSCs and their recruitment to sites of injury, inflammation, or other pathologic perturbations.

Recent epidemiological studies have shown that inflammatory bowel disease is associated with periodontal disease. The oral-gut microbiota axis is a potential mechanism intersecting the two diseases. Porphyromonas gingivalis is currently considered a keystone oral pathogen involved in periodontal disease pathogenesis and disease progression. Recent studies have shown that oral ingestion of P. gingivalis leads to intestinal inflammation. However, the molecular underpinnings of P. gingivalis-mediated gut inflammation have remained elusive. In this study, we show that the oral administration of P. gingivalis indeed leads to ileal inflammation and alteration in gut microbiota with significant reduction in bacterial alpha diversity despite the absence of P. gingivalis in the lower gastrointestinal tract. Utilizing an antibiotic-conditioned mouse model, cecal microbiota transfer experiments were performed to demonstrate that P. gingivalis-induced dysbiotic gut microbiota is sufficient to reproduce gut pathology. Furthermore, we observed a significant expansion in small intestinal lamina propria IL9(+) CD4(+) T cells, which was negatively correlated with both bacterial and fungal alpha diversity, signifying that P. gingivalis-mediated intestinal inflammation may be due to the subsequent loss of gut microbial diversity. Finally, we detected changes in gene expression related to gut epithelial barrier function, showing the potential downstream effect of intestinal IL9(+)CD4(+) T-cell induction. This study for the first time showed the mechanism behind P. gingivalis-mediated intestinal inflammation where P. gingivalis indirectly induces intestinal IL9(+) CD4(+) T cells and inflammation by altering the gut microbiota. Understanding the mechanism of P. gingivalis-mediated intestinal inflammation may lead to the development of novel therapeutic approaches to alleviate the morbidity from inflammatory bowel disease patients with periodontal disease.

Although there is a clear relationship between the degree of obesity and periodontal disease incidence, the mechanisms that underpin the links between these conditions are not completely understood. Understanding that myeloid-derived suppressor cells (MDSCs) are expanded during obesity and operate in a context-defined manner, we addressed the potential role of MDSCs to contribute toward obesity-associated periodontal disease. Flow cytometry revealed that in the spleen of mice fed a high-fat diet (HFD), expansion in monocytic MDSCs (M-MDSCs) significantly increased when compared with mice fed a low-fat diet (LFD). In the osteoclast differentiation assay, M-MDSCs isolated from the bone marrow of HFD-fed mice showed a larger number and area of osteoclasts with a greater number of nuclei. In the M-MDSCs of HFD-fed mice, several osteoclast-related genes were significantly elevated when compared with LFD-fed mice according to a focused transcriptomic platform. In experimental periodontitis, the number and percentage of M-MDSCs were greater, with a significantly larger increase in HFD-fed mice versus LFD-fed mice. In the spleen, the percentage of M-MDSCs was significantly higher in HFD-fed periodontitis-induced (PI) mice than in LFD-PI mice. Alveolar bone volume fraction was significantly reduced in experimental periodontitis and was further decreased in HFD-PI mice as compared with LFD-PI mice. The inflammation score was significantly higher in HFD-PI mice versus LFD-PI mice, with a concomitant increase in TRAP staining for osteoclast number and area in HFD-PI mice over LFD-PI mice. These data support the concept that M-MDSC expansion during obesity to become osteoclasts during periodontitis is related to increased alveolar bone destruction, providing a more detailed mechanistic appreciation of the interconnection between obesity and periodontitis.