Single‐cell multi‐omics analysis presents the landscape of peripheral blood T‐cell subsets in human chronic prostatitis/chronic pelvic pain syndrome

Cumulative evidence suggests that abnormal differentiation of T lymphocytes influences the pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Thus, understanding the immune activation landscape of CP/CPPS would be helpful for improving therapeutic strategies. Here, we utilized BD™ AbSeq to digitally quantify both the protein and mRNA expression levels in single peripheral blood T cells from two CP/CPPS patients and two healthy controls. We utilized an integrated strategy based on canonical correlation analysis of 10 000+ AbSeq profiles and identified fifteen unique T‐cell subpopulations. Notably, we found that the proportion of cluster 0 in the CP/CPPS group (30.35%) was significantly increased compared with the proportion in the healthy control group (9.38%); cluster 0 was defined as effector T cells based on differentially expressed genes/proteins. Flow cytometry assays confirmed that the proportions of effector T‐cell subpopulations, particularly central memory T cells, T helper (Th)1, Th17 and Th22 cells, in the peripheral blood mononuclear cell populations of patients with CP/CPPS were significantly increased compared with those of healthy controls (P < 0.05), further confirming that aberration of effector T cells possibly leads to or intensifies CP/CPPS. Our results provide novel insights into the underlying mechanisms of CP/CPPS, which will be beneficial for its treatment.     

Cancer‐associated mutations in VAV1 trigger variegated signaling outputs and T‐cell lymphomagenesis

Mutations in VAV1, a gene that encodes a multifunctional protein important for lymphocytes, are found at different frequencies in peripheral T‐cell lymphoma (PTCL), non‐small cell lung cancer, and other tumors. However, their pathobiological significance remains unsettled. After cataloguing 51 cancer‐associated VAV1 mutations, we show here that they can be classified in five subtypes according to functional impact on the three main VAV1 signaling branches, GEF‐dependent activation of RAC1, GEF‐independent adaptor‐like, and tumor suppressor functions. These mutations target new and previously established regulatory layers of the protein, leading to quantitative and qualitative changes in VAV1 signaling output. We also demonstrate that the most frequent VAV1 mutant subtype drives PTCL formation in mice. This process requires the concurrent engagement of two downstream signaling branches that promote the chronic activation and transformation of follicular helper T cells. Collectively, these data reveal the genetic constraints associated with the lymphomagenic potential of VAV1 mutant subsets, similarities with other PTCL driver genes, and potential therapeutic vulnerabilities.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

Architecture of the active post‐translational Sec translocon

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.     

ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.     

DJ‐1 depletion prevents immunoaging in T‐cell compartments

Decline in immune function during aging increases susceptibility to different aging‐related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T‐cell subpopulations, still remain largely elusive. Here, we show that loss of DJ‐1 encoded by PARK7/DJ‐1, causing early‐onset familial Parkinson’s disease (PD), unexpectedly diminished signs of immunoaging in T‐cell compartments of both human and mice. Compared with two gender‐matched unaffected siblings of similar ages, the index PD patient with DJ‐1 deficiency showed a decline in many critical immunoaging features, including almost doubled non‐senescent T cells. The observation was further consolidated by the results in 45‐week‐old DJ‐1 knockout mice. Our data demonstrated that DJ‐1 regulates several immunoaging features via hematopoietic‐intrinsic and naïve‐CD8‐intrinsic mechanisms. Mechanistically, DJ‐1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T‐cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ‐1 in regulating immunoaging, discovering a potent target to interfere with immunoaging‐ and aging‐associated diseases.     

Advances in single‐cell sequencing and its application to musculoskeletal system research

In recent years, single‐cell sequencing (SCS) technologies have continued to advance with improved operating procedures and reduced cost, leading to increasing practical adoption among researchers. These emerging technologies have superior abilities to analyse cell heterogeneity at a single‐cell level, which have elevated multi‐omics research to a higher level. In some fields of research, application of SCS has enabled many valuable discoveries, and musculoskeletal system offers typical examples. This article reviews some major scientific issues and recent advances in musculoskeletal system. In addition, combined with SCS technologies, the research of cell or tissue heterogeneity in limb development and various musculoskeletal system clinical diseases also provides new possibilities for treatment strategies. Finally, this article discusses the challenges and future development potential of SCS and recommends the direction of future applications of SCS to musculoskeletal medicine.     

Liposomal phytohemagglutinin: In vivo T‐cell activator as a novel pan‐cancer immunotherapy

Immunotherapy is an attractive approach for treating cancer. T‐cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T‐cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T‐cell exhaustion.     

Identification of necroptosis‐related gene signature and characterization of tumour microenvironment infiltration in non‐small‐cell lung cancer

Necroptosis is a programmed necrosis in a caspase‐independent fashion. The role of necroptosis‐related genes (NRGs) in lung cancer remains unknow. Herein, we classified TCGA‐LUAD cohort into two necroptosis‐related subtypes (C1 and C2) by consensus clustering analysis. The result showed that subtype C1 had a favourable prognosis and higher infiltration levels of immune cells. Moreover, subtype C1 was more activated in immune‐associated pathways. Then, we established an NRG prognosis model (NRG score) composed of six NRGs (RIPK3, MLKL, TLR2, TLR4, TNFRSF1A, NDRG2) and divided the cohort into low‐ and high‐risk group. We found that the NRG score was associated with prognosis, tumour immune microenvironment and tumour mutation burden. We also constructed an accurate nomogram model to improve the clinical applicability of NRG score. The result indicated that NRG score may be an independent prognostic marker for lung cancer patients. Taken together, we established a prognosis model that may deepen the understanding of NRGs in lung cancer and provide a basis for developing more effective immunotherapy strategies.     

Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19

SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses.     

MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.     

Sparstolonin B suppresses free fatty acid palmitate‐induced chondrocyte inflammation and mitigates post‐traumatic arthritis in obese mice

Abnormal lipid metabolism, such as systemic increased free fatty acid, results in overproduction of pro‐inflammatory enzymes and cytokines, which is crucial in the development of obesity‐related osteoarthritis (OA). However, there are only a few drugs that target the lipotoxicity of OA. Recent researches have documented that the traditional Chinese medicine, Sparstolonin B (Ssn B), exerted anti‐inflammatory effects in various diseases, but not yet in OA. On the basis of this evidence, our works purposed to evaluate the effect of Ssn B on free fatty acid (FFA) palmitate (PA)‐stimulated human osteoarthritic chondrocytes and obesity‐associated mouse OA model. We found that Ssn B suppressed PA‐triggered inflammatory response and extracellular matrix catabolism in a concentration‐dependent approach. In vivo, Ssn B treatment inhibited cartilage degeneration and subchondral bone calcification caused by joint mechanical imbalance and alleviated metabolic inflammation in obesity. Mechanistically, co‐immunoprecipitine and molecular docking analysis showed that the formation of toll­like receptor 4 (TLR4)/myeloid differentiation protein‐2 (MD‐2) complex caused by PA was blocked by Ssn B. Subsequently, it leads to inactivation of PA‐caused myeloid differentiation factor 88 (MyD88)‐dependent nuclear factor‐kappaB (NF‐κB) cascade. Together, these findings demonstrated that Ssn B is a potential treatment agent for joint degenerative diseases in obese individuals.     

Phospho‐regulation,nucleotide binding and ion access control in potassium‐chloride cotransporters

Potassium‐coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho‐regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo‐EM structures of human KCC3b and KCC1, revealing structural determinants for phospho‐regulation in both N‐ and C‐termini. We show that phospho‐mimetic KCC3b is arrested in an inward‐facing state in which intracellular ion access is blocked by extensive contacts with the N‐terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho‐regulatory site in the KCC1 N‐terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP‐binding pocket in the large C‐terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.     

Application of T‐cell receptor repertoire as a novel monitor in dynamic tracking and assessment: A cohort‐study based on RA patients

T‐cell receptor repertoire (TCRR) sequencing has been widely applied in many fields as a novel tool. This study explored characteristics of TCRR in detail with a cohort of 598 rheumatoid arthritis (RA) patients before and after anti‐rheumatic treatments. We highlighted the abnormal TCRR distribution in RA characterized by decreased diversity and increased proportion of hyperexpanded clones (HECs), which was potentially attributed to skewed usage of global V/J segments but not a few certain ones. Enriched motifs analysis in RA community demonstrated the huge heterogeneity of CDR3 sequences, so that individual factors are strongly recommended to be taken into consideration when it comes to clinical application of TCRR. Disease‐modifying antirheumatic drugs (DMARDs) can regulate immune system through recovery of TCRR richness to relieve symptoms. Remarkably, sensitive gene profile and advantageous gene profile were identified in this study as new biomarkers for different DMARDs regimens.     

6‐Monoacetylmorphine‐antibody distribution in tissues from heroin‐related death cases: An experimental study to investigate the distributive response

Heroin, a semisynthetic opioid drug synthesized from morphine, is the 3,6‐diacetyl ester of morphine (diacetylmorphine). The post‐mortem diagnosis of heroin‐related death could be an issue and usually rely on a combination of investigations, including the autopsy, histological and toxicological analysis. We conducted the present study to evaluate the correlation between the heroin concentration in biological fluids (peripheral blood, bile and urine) and the post‐mortem anti‐6‐MAM antibody expression in various tissues (brain, heart, lung, liver and kidney) using immunohistochemical staining. A quantitative analysis of the immunohistochemical reaction was carried out. 45 cases of heroin‐related death investigated at the Forensic Pathology Institutes of the University of Rome, Foggia and Pisa were included. The control group was composed of 15 cases of death due to other causes, without brain lesions and negative toxicological analysis for drugs. We found a positive immunohistochemical reaction in different organs and it was related to the timing of heroin metabolization. No reaction was found in the control group. Our findings show that immunohistochemistry can be a valuable tool for the post‐mortem diagnosis of acute heroin abuse. A better understanding of the timing of heroin''s metabolism can be useful in the forensic field and for future therapeutic applications.     

20‐HETE synthesis inhibition attenuates traumatic brain injury–induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC‐1α pathway: A translational study

Objectives20‐hydroxyeicosatetraenoic acid (20‐HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20‐HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms.Materials and methodsNeurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post‐TBI effects of HET0016, an inhibitor of 20‐HETE synthesis, and their underlying mechanisms.ResultsThe level of 20‐HETE was found to be increased significantly after TBI in mice. 20‐HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post‐TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC‐1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC‐1α and the abnormal mitochondrial dynamics induced by 20‐HETE in vitro. Furthermore, plasma 20‐HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score.ConclusionsThe inhibition of 20‐HETE synthesis represents a novel strategy to mitigate TBI‐induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC‐1α pathway.     

The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c‐di‐GMP network

Bacteria use signal transduction systems to sense and respond to their external environment. The two‐component system CpxA/CpxR senses misfolded envelope protein stress and responds by up‐regulating envelope protein factors and down‐regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS‐inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c‐di‐GMP regulators were also up‐regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c‐di‐GMP phenotypes in biofilm formation and swimming. Increased production of cellular c‐di‐GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c‐di‐GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.