CXCR4 Expression Research Articles

Time-dependent phenotypical changes of microglia drive alterations in hippocampal synaptic transmission in acute slices

It is widely acknowledged that microglia actively regulate synaptic function in the brain. Remarkably, much of our understanding regarding the role of microglia in synaptic regulation is derived from studies in acute brain slices. However, it is still uncertain to what extent the preparation and maintenance of acute slices can influence microglial function and whether microglial changes may affect synaptic transmission. In this study, we examined the impact of acute slice resting time on hippocampal CA1 microglia, by assessing morphological and functional parameters at two distinct time intervals. We report that after 4 h from slicing microglia undergo morphological, functional, and transcriptional changes, including a decrease in the number of branches and in their movement speed. Furthermore, microglia acquire a reactive phenotype, characterized by increased amplitude of outward rectifying K+ currents, increased expression of the pro-inflammatory cytokine Tnfα and altered expression of the microglial receptors Cx3cr1 and P2y12r. We also examined time-dependent changes of excitatory synaptic transmission in CA1 pyramidal neurons from acute hippocampal slices, reporting time-dependent decrease in both amplitude and frequency of postsynaptic currents (sEPSCs), along with a decrease in spine density. Noticeably, sEPSCs amplitude decrease was absent in slices prepared from PLX5622 microglia-depleted mice, suggesting that this time-dependent effect on synaptic transmission is microglia-dependent. Our findings highlight possible causal relation between microglia phenotypic changes in the hours following slice preparation and concomitant synaptic changes, pointing to the mechanisms of acute synaptic modulation, whose understanding is crucial for unraveling microglia-neurons interplay in nature. Furthermore, they emphasize the potential issues associated with experimental time windows in ex vivo samples.

Stearoyl-CoA desaturase in CD4+ T cells suppresses tumor growth through activation of the CXCR3/CXCL11 axis in CD8+ T cells.

Within the tumor microenvironment, altered lipid metabolism promotes cancer cell malignancy by activating oncogenic cascades; however, impact of lipid metabolism in CD4+ tumor-infiltrating lymphocytes (TILs) remains poorly understood. Here, we elucidated that role of stearoyl-CoA desaturase (SCD) increased by treatment with cancer-associated fibroblast (CAF) supernatant in CD4+ T cells on their subset differentiation and activity of CD8+ T cells. In our study, we observed that CD4+ TILs had higher lipid droplet content than CD4+ splenic T cells. In tumor tissue, CAF-derived supernatant provided fatty acids to CD4+ TILs, which increased the expression of SCD and oleic acid (OA) content. Increased SCD expression by OA treatment enhanced the levels of Th1 cell markers TBX21, interleukin-2, and interferon-γ. However, SCD inhibition upregulated the expression of regulatory T (Treg) cell markers, FOXP3 and transforming growth factor-β. Comparative fatty acid analysis of genetically engineered Jurkat cells revealed that OA level was significantly higher in SCD-overexpressing cells. Overexpression of SCD increased expression of Th1 cell markers, while treatment with OA enhanced the transcriptional level of TBX21 in Jurkat cells. In contrast, palmitic acid which is higher in SCD-KO cells than other subclones enhanced the expression of Treg cell markers through upregulation of mitochondrial superoxide. Furthermore, SCD increased the secretion of the C-X-C motif chemokine ligand 11 (CXCL11) from CD4+ T cells. The binding of CXCL11 to CXCR3 on CD8+ T cells augmented their cytotoxic activity. In a mouse tumor model, the suppressive effect of CD8+ T cells on tumor growth was dependent on CXCR3 expression. These findings illustrate that SCD not only orchestrates the differentiation of T helper cells, but also promotes the antitumor activity of CD8+ T cells, suggesting its function in adverse tumor microenvironments.

A Small Molecule Antagonist of CX3CR1 (KAND567) Inhibited the Tumor Growth-Promoting Effect of Monocytes in Chronic Lymphocytic Leukemia (CLL)

Background/Objectives: Nurse-like cells (NLCs) derived from monocytes in the tumor microenvironment support the growth of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the effects of a CX3CR1 (fractalkine receptor) antagonist (KAND567) on autologous monocytes and their pro-survival effects on CLL cells in vitro. Methods: Plasma concentration of CX3CL1 was determined by ELISA and CX3CR1 expression by flow cytometry. CD19+ cells and autologous monocytes from patients with CLL and healthy donors were treated with KAND567 either in co-culture or alone. The apoptosis of CD19+ cells and monocytes was determined by Annexin V/PI staining and live-cell imaging. Results: Plasma concentration of CX3CL1 (fractalkine) was significantly higher in patients with CLL (n = 88) than in healthy donors (n = 32) (p < 0.0001), with higher levels in patients with active compared to non-active disease (p < 0.01). CX3CR1 was found on monocytes but not B cells in patients and controls. Levels of intermediate and non-classical CX3CR1+ monocytes were higher in patients with CLL than in controls (p < 0.001), particularly in those with active disease (p < 0.0001). Co-culture experiments revealed that autologous monocytes promoted the survival of both malignant and normal B cells and that KAND567 selectively inhibited the growth of CLL cells in a dose-dependent manner but only in the presence of autologous monocytes (p < 0.05). Additionally, KAND567 inhibited the transition of monocytes to NLCs in CLL (p < 0.05). Conclusions: Our data suggest that the CX3CR1/CX3CL1 axis is activated in CLL and may contribute to the NLC-driven growth-promoting effects of CLL cells. KAND567, which is in clinical trials in other disorders, should also be explored in CLL.

DNAR-01. THERAPEUTIC APPLICATIONS OF MESENCHYMAL STEM CELL LOADED WITH GOLD NANOPARTICLES FOR REGENERATIVE MEDICINE

Abstract In the present study, the various concentrations of AuNP (1.25, 2.5, 5, 10 ppm) were prepared to investigate the biocompatibility, biological performances and cell uptake efficiency via Wharton’s jelly mesenchymal stem cells and rat model. The pure AuNP, AuNP combined with Col (AuNP-Col) and FITC conjugated AuNP-Col (AuNP-Col-FITC) were characterized by Ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and Dynamic Light Scattering (DLS) assays. For in vitro examinations, we explored whether the Wharton’s jelly MSCs had better viability, higher CXCR4 expression, greater migration distance and lower apoptotic-related proteins expression with AuNP 1.25 and 2.5 ppm treatments. Furthermore, we considered whether the treatments of 1.25 and 2.5 ppm AuNP could induce the CXCR4 knocked down Wharton’s jelly MSCs to express CXCR4 and reduce the expression level of apoptotic proteins. We also treated the Wharton’s jelly MSCs with AuNP-Col to investigate the intracellular uptake mechanisms. The evidence demonstrated the cells uptake AuNP-Col through clathrin-mediated endocytosis and the vacuolar-type H+-ATPase pathway with good stability inside the cells to avoid lysosomal degradation as well as better uptake efficiency. Additionally, the results from in vivo examinations elucidated the 2.5 ppm of AuNP attenuated foreign body responses and had better retention efficacy with tissue integrity in animal model. In conclusion, the evidence demonstrates that AuNP shows promise as a biosafe nanodrug delivery system for development of regenerative medicine coupled with Wharton’s jelly MSCs.

Modeling Lymphoma Angiogenesis, Lymphangiogenesis, and Vessel Co-Option, and the Effects of Inhibition of Lymphoma–Vessel Interactions with an αCD20-EndoP125A Antibody Fusion Protein

Lymphoma growth, progression, and dissemination require tumor cell interaction with supporting vessels and are facilitated through tumor-promoted angiogenesis, lymphangiogenesis, and/or lymphoma vessel co-option. Vessel co-option has been shown to be responsible for tumor initiation, metastasis, and resistance to anti-angiogenic treatment but is largely uncharacterized in the setting of lymphoma. We developed an in vitro model to study lymphoma–vessel interactions and found that mantle cell lymphoma (MCL) cells co-cultured on Matrigel with human umbilical vein (HUVEC) or human lymphatic (HLEC) endothelial cells migrate to and anneal with newly formed capillary-like (CLS) or lymphatic-like (LLS) structures, consistent with lymphoma–vessel co-option. To inhibit this interaction, we constructed an antibody fusion protein, αCD20-EndoP125A, linking mutant anti-angiogenic endostatin (EndoP125A) to an αCD20-IgG1-targeting antibody. αCD20-EndoP125A inhibited both CLS and LLS formation, as well as MCL migration and vessel co-option. Lymphoma vessel co-option requires cell migration, which is regulated by chemokine–chemokine receptor interactions. CXCL12 and its receptor, CXCR4, are highly expressed by both endothelial cells forming CLS and by MCL cells during vessel co-option. αCD20-EndoP125A suppressed expression of both CXCL12 and CXCR4, which were required to facilitate CLS assembly and vessel co-option. We also tested αCD20-EndoP125A effects in vivo using an aggressive murine B cell lymphoma model, 38c13-hCD20, which demonstrated rapid growth and dissemination to tumor-draining lymph nodes (TDLNs) and the spleen, lung, and brain. The pattern of lymphoma distribution and growth within the lung was consistent with vessel co-option. As predicted by our in vitro model, αCD20-EndoP125A treatment inhibited primary tumor growth, angiogenesis, and lymphangiogenesis, and markedly reduced the number of circulating tumor cells and lymphoma dissemination to TDLNs and the lungs, spleen, and brain. αCD20-EndoP125A inhibited lymphoma vessel co-option within the lung. Marked inhibition of MCL primary tumor growth and dissemination were also seen using an MCL xenograft model. The ability of αCD20-EndoP125A to inhibit angiogenesis, lymphangiogenesis, and lymphoma vessel co-option provides a novel therapeutic approach for inhibition of lymphoma progression and dissemination.

Chemokine receptor CXCR7 antagonism ameliorates cardiac and renal fibrosis induced by mineralocorticoid excess

Cardiorenal fibrosis is a common feature of chronic cardiovascular disease and recent data suggests that cytokines and chemokines may also drive fibrosis. Here we tested the hypothesis that CXCR7, a highly conserved chemokine receptor, contributes to cardiac and renal fibrosis. We generated an anti-mouse CXCR7-specific monoclonal antibody (CXCR7 mAb) and tested its anti-fibrotic actions in cardiorenal fibrosis induced using the deoxycorticosterone acetate/uni-nephrectomy (DOCA-UNX) model. CXCR7 mAb treatment (10 mg/kg, twice weekly for 6 weeks) significantly attenuated the development of cardiac and renal fibrosis, and reduced fibrotic and inflammatory gene expression levels, in the absence of an effect on blood pressure. Immunohistochemical analysis demonstrated an increase in the vascular expression of CXCR7 in DOCA-UNX-treated mice. This study demonstrated that a CXCR7 mediated pathway plays a significant role in cardiac and renal fibrosis induced by DOCA-UNX treatment. Accordingly, antagonism of CXCR7 may provide a therapeutic opportunity to mitigate against fibrosis in the setting of mineralocorticoid excess.

In Vivo Detection of Lymph Nodes Metastasis of ESCC Using CXCR4-Targeted Tracer [64Cu]Cu-NOTA-CP01.

Esophageal squamous cell carcinoma (ESCC) frequently exhibits skip metastasis to lymph nodes; however, non-invasive imaging techniques capable of directly visualizing metastatic lymph nodes (MLN) are still lacking. Although biopsy is the clinical standard method, it is invasive and poses risks to patient health. This study aims to detect MLN in an intralymphatic tumor metastasis model of ESCC using the CXCR4-targeted tracer [64Cu]Cu-NOTA-CP01. The CXCR4 expression in ESCC cell lines was assessed using Western blot and immunofluorescence. An intralymphatic tumor metastasis model was established and monitored using bioluminescence imaging (BLI). Small animal PET studies and biodistribution studies were performed to evaluate the specificity of [64Cu]Cu-NOTA-CP01 for MLN. Histopathology evaluation was employed to check for the presence of metastatic tumor cells and to assess CXCR4 expression levels in the metastatic lymph nodes. The intralymphatic tumor metastasis model was successfully established using the EC109/Luc cell line, which exhibited high CXCR4 expression, as verified by BLI. PET/CT imaging showed that the MLN uptakes in the baseline group were significantly inhibited in the blocking group. The ratios of MLN/muscle and MLN/blood werealso significantly higher in the baseline group than in the blocking group. Ex vivo PET/CT imaging of MLN corroborated the in vivo data. Biodistribution studies further supported the PET imaging studies, showing rapid clearance of the tracer from the blood and major organs, with significantly higher MLN/muscle and MLN/blood ratios in the baseline group compared to the blocking group. Histopathological staining verified positive CXCR4 expression in these lymph nodes containing metastatic tumor cells. Targeting CXCR4 with [64Cu]Cu-NOTA-CP01 for PET imaging of lymph nodes metastasis represents a promising approach that warrants further investigation. These findings have the potential to enhance diagnostic and therapeutic strategies for individuals with lymph nodes metastasis of ESCC.

Zoledronate interrupts pre-osteoclast-induced angiogenesis via SDF-1/CXCR4 pathway

IntroductionIn this study, we tested the hypothesis that pre-osteoclast signaling is key in triggering post-traumatic angiogenesis in alveolar bone via the SDF-1/CXCR4 pathway. Interruption of osteoclast differentiation through zoledronate (Zol) disrupts the crosstalk between pre-osteoclasts and endothelial cells, hindering the initial angiogenic reaction following dental trauma. This disruption could therefore play a role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). MethodsThe effect of zoledronate on the expression of SDF1 was tested in pre-osteoclasts (POC) in vitro. Then, we tested the effect of pre-osteoclast conditioned medium on HUVEC cell differentiation, migration, tube-formation, and CXCR4 expression and activity in-vitro. Lastly, we quantified the effect of zoledronate treatment on post-traumatic vascular perfusion of alveolar bone, using microCT-angiography and immunohistochemistry. ResultsSDF-1 mRNA expression decreased in Zol-treated POCs (p = 0.02). Flow-Cytometry analysis showed a decrease in CXCL-12+ (SDF-1α) expressing POCs with Zol treatment (p = 0.0058). On the other hand, CXCR4 mRNA expression was significantly inhibited in Zol-treated HUVECs (p = 0.0063). CXCR4 protein expression and activity showed a corresponding dose-dependent downregulation HUVEC surface treated with conditioned media from POC treated with Zol (p = 0.008 and 0.03, respectively). Similar inhibition was observed of HUVEC migration (p = 0.0012), and tube formation (p < 0.0001), effects that were reversed with SDF-1. Finally, there was a significant reduction of CD31+ HUVECs in Alveolar bone of Zol-treated rats (p = 0.0071), confirmed by significantly lower percentage of blood vessel volume (p = 0.026), and marginally lower vessel number (p = 0.062) in the alveolar bone. ConclusionPre-osteoclasts play a crucial role in the initial angiogenic response in alveolar bone following dental extraction. Disruption of this process may be a predisposing factor to osteonecrosis.

Exosomal miR-25 from Mesenchymal stem cells inhibits T cells migration and Alleviates Type 1 diabetes mellitus by Targeting CXCR3 models.

Mesenchymal stem cells (MSCs) have demonstrated promising therapeutic potential in the treatment of type 1 diabetes mellitus (T1DM); however, the underlying mechanism remains unclear. The primary pathological mechanism of T1DM involves activated T cells infiltrating the pancreas, leading to islet inflammation and the destruction of β-cells. However, the question of whether exosomes derived from MSCs can suppress the migration of T cells to the pancreas in the context of T1DM remains unresolved. In this study, we observed that miR-25 was highly expressed in MSCs exosomes and associated with signaling pathways related to cell migration. In vitro assay, we synthesized a miR-25 mimic and transiently transfected it into activated T cells, which revealed that miR-25 can effectively reduce the expression of CXCR3. Additionally, according to the in vivo T1DM mouse model, we found that there was a significant increase in miR-25 levels in T1DM mice treated with MSCs and the number of T cells decreased. Overall, our findings suggest that MSCs exosomes containing miR-25 can impede the infiltration of activated T cells into the pancreas in T1DM by repressing CXCR3 expression in these cells.

Pulse Activation of Retinoic Acid Receptor Enhances Hematopoietic Stem Cell Homing by Controlling CXCR4 Membrane Presentation.

The interplay between metabolic signaling and stem cell biology has gained increasing attention, though the underlying molecular mechanisms remain incompletely elucidated. In this study, we identify and characterize the role of adapalene (ADA), a retinoic acid receptor (RAR) agonist, in modulating the migration behavior of hematopoietic stem cells (HSCs). Our initial findings reveal that ADA treatment suppresses hematopoietic stem and progenitor cell (HSPC) mobilization induced by AMD3100 and G-CSF. Furthermore, we demonstrate that ADA treatment upregulates the surface expression of CXCR4 on HSPCs, resulting in enhanced chemotaxis towards CXCL12. Mechanistically, our study suggests that ADA enhances CXCR4 surface presentation without increasing CXCR4 mRNA levels, pointing towards a non-canonical role of RAR signaling in regulating intracellular trafficking of CXCR4. In vivo experiments show that ADA administration significantly enhances HSC homing efficiency. Additionally, competitive transplantation assays indicate a marked increase in donor chimerism following ADA treatment. These findings highlight the critical role of retinoic acid signaling in regulating HSC homing and suggest its potential for advancing novel HSC-based therapeutic strategies.

Local delivery of soluble fractalkine (CX3CL1) peptide restores ribbon synapses after noise-induced cochlear synaptopathy.

Cochlear ribbon synapses between sensory inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are vulnerable to rapid and primary damage and/or loss due to noise overexposure. Such damaged ribbon synapses can repair spontaneously in mouse and guinea pig. However, the mechanisms for synaptic repair are unclear. Previously, we have demonstrated a critical role for the fractalkine signaling axis (CX3CL1-CX3CR1) in synaptic repair, wherein noise-damaged ribbon synapses are spontaneously repaired in the presence of fractalkine receptor (CX3CR1) expressed by cochlear macrophages. Here, we examined whether local administration of chemokine fractalkine ligand (CX3CL1 or FKN) in the form of a peptide is effective in restoring synapses and hearing loss after noise-induced cochlear synaptopathy (NICS). Specifically, the efficacy of different isoforms of FKN was evaluated for restoration of loss of IHC ribbon synapses and hearing after NICS. A single transtympanic injection of soluble isoform of FKN (sFKN) peptide at 1 day after synaptopathic noise trauma for 2 hours at 93 decibel sound pressure level led to significant recovery of auditory brainstem response (ABR) thresholds, ABR peak I amplitudes and ribbon synapses in FKN knockout mice when compared to mice injected with membrane-bound FKN peptide (mFKN). Likewise, local treatment with sFKN peptide in FKN wild type mice restored synaptopathic noise-damaged ribbon synapses and ABR peak I amplitudes. Mechanistically, FKN regulates macrophage numbers in the damaged cochlea and in the absence of macrophages, sFKN failed to restore loss of synapses and hearing after NICS. Furthermore, sFKN treatment attenuated cochlear inflammation after NICS without altering the expression of CX3CR1. Finally, injected sFKN peptide was detectable inside the cochlea for 24 h localized to the basilar membrane and spiral lamina near the sensory epithelium. These data provide a proof-of-principle that local delivery of an immune factor, sFKN is effective in restoring ribbon synapses and hearing loss after NICS in a macrophage-dependent manner and highlights the potential of sFKN as an immunotherapy for cochlear synaptopathy due to noise.

The myocardium of human dilated nonischemic cardiomyopathy harbors unique tissue-resident lymphocyte subsets

Abstract Purpose The etiology of dilated nonischemic cardiomyopathy (NICM) is often unclear, but thought to be multifactorial, involving an interplay between genetic factors and direct myocardial damage caused by infection, autoimmunity, or toxins. Tissue-resident lymphocytes are non-circulating immune cells which are retained within various tissues and play integral roles in maintaining homeostasis, responding to infection, regulating inflammation, and mediating tissue repair. Despite their likely involvement in various cardiac pathologies, the presence of tissue-resident lymphocytes in the human heart has not yet been well-characterized, neither in health nor in disease. Methods Human left ventricular tissue and paired peripheral blood samples were obtained from six adult organ donors with normal heart function who died of noncardiac causes and from the explants of six adult patients with end-stage nonischemic cardiomyopathy. Heart tissue was processed using mechanical and enzymatic digestion. Mononuclear cells were isolated from both heart and paired blood by density centrifugation. Isolated mononuclear cells were surface stained with an antibody cocktail and interrogated using multidimensional flow cytometry. Results The adult human heart contains a diverse immune cell population (Fig 1A). Both healthy and NICM cardiac lymphocytes express distinct phenotypic profiles which resemble those of resident lymphocytes found across various tissues, distinct from those isolated from paired blood. Frequencies of effector memory T (Tem) cells (CD45RA- CCR7-) were higher in heart than in blood among both CD4+ and CD8+ T-cells. In heart compared to blood, significantly more Tem expressed CD69, a canonical marker of tissue-resident memory T (Trm) cells (Fig 1B). Cardiac Trm, especially CD8+ Trm, expressed classical Trm signature markers including integrins CD103 (epithelial binding) and CD49a (collagen binding). NICM Trm expressed more CD49a than healthy heart Trm (Fig 1C). NK-cells also demonstrated higher CD69 expression (trNK) in heart than in blood, with associated expression of CD103, CD49a, and CXCR6 (Fig 1D). Compared to healthy hearts, NICM trNK were predominantly CD56bright/CD16- (Fig 1E and 1F) and expressed less CD57 (Fig 1D), overall indicating a less mature state with lower cytotoxic potential. Confocal imaging of cardiac tissue revealed CD69-expressing tissue-resident lymphocytes within the cardiac interstitium. Conclusions The adult human heart contains diverse populations of lymphocytes which express canonical markers of tissue residency, similar to lymphocytes resident in other tissues. Compared to healthy hearts, NICM hearts have more CD49a+ Trm, perhaps related to greater fibrosis, and also harbor a distinct subset of immature tissue-resident NK cells which may shed light on pathophysiology. Future work is focused on better exploring the functional phenotypes of these resident cells and better localizing them within the myocardium.FA - Figure 1

Inhibition of fractalkine signalling in patients with acute myocardial infarction - First results from the FRACTAL trial

Abstract Background We have previously demonstrated a strong association between expression of the fractalkine receptor CX3CR1 on lymphocytes and microvascular obstruction (MVO) in patients undergoing primary percutaneous coronary intervention (PPCI) for ST-elevation myocardial infarction. Our pre-clinical studies also found an improvement in infarct size, inflammation and intramyocardial haemorrhage (IMH) when rats were pre-treated with the fractalkine inhibitor KAND567. Purpose FRACTAL (FRACTalkine inhibition in Acute myocardial infarction) study was a phase IIa, pilot, randomized, 2-arm parallel group, placebo-controlled, double blinded, multi-centre trial. Its primary aim was to assess the safety and tolerability of intravenous and oral administration of the fractalkine inhibitor KAND567 as well as reducing signs of reperfusion injury in STEMI patients undergoing PPCI. Methods Patients with a new diagnosis of anterior STEMI, who had index chest pain within 5 hours and angiographic occlusion of the LAD, were randomised on a 1:1 basis to receive either KAND567 or placebo over a period of 72 hours. All patients then underwent a baseline MRI scan at 3 days with a follow-up scan at 3 months, paralleled by longitudinal deep immuno­phenotyping over 9 time points. Results A total of 71 patients were enrolled into the study with 64 (90.1%) being male patients. The mean age of patients was 59.2 years. 37 patients received KAND567, whilst 34 received placebo over 72-hour period. Throughout the 3-month follow-up period, 23 (62%) patients from the KAND567 arm as compared to 24 (71%) patients in the placebo arm had reported adverse events. There was no statistical difference in serious adverse events (SAEs) between both arms with 9 documented in the placebo arm and 12 in the KAND567 arm. Safety bloods also demonstrated no significant differences between the two groups across all time points. There was a higher incidence of left ventricular (LV) thrombus in the placebo arm as compared to the KAND567 arm (17.6% vs. 2.7%; p=0.049). Additionally, the study also demonstrated a lower occurrence of IMH in the KAND567 arm compared to the placebo arm (35.5% vs. 55.2%; p=0.19). Deep immunephenotyping suggested clear target engagement in the KAND567 arm in natural killer cells as well as in cytotoxic T-lymphocytes. Conclusion This first in-man study demonstrates that inhibition of the fractalkine pathway in STEMI patients appears safe and well-tolerated, while demonstrating a promising potential in preventing LV thrombus and IMH. The results suggest a new mechanism and class of drug to target reperfusion injury, clearly warranting a larger phase III study.LV thrombus and IMH

Causality of immune cells and endometriosis: a bidirectional mendelian randomization study

BackgroundEndometriosis, a prevalent chronic condition, afflicts approximately 10% of women in their reproductive years. Emerging evidence implicates immune cells in the pathogenesis of endometriosis, particularly in angiogenesis, tissue proliferation, and lesion invasion. This investigation employs two-sample Mendelian Randomization (MR) to dissect the bidirectional causal relationships between immune cell profiles and endometriosis.MethodsWe leveraged publicly available genome-wide association study (GWAS) data to elucidate the causal interplay between immune cell traits and endometriosis. Utilizing GWAS summary statistics ranging from accession numbers GCST90001391 to GCST90002121 and endometriosis data from the FinnGen study GWAS (8,288 endometriosis cases and 68,969 controls), we adopted stringent criteria for instrumental variable selection. We applied MR-Egger, weighted median, inverse variance weighted (IVW), and weighted mode methods to derive causal estimates. To address potential heterogeneity and pleiotropy, Cochran’s Q test, MR-Egger intercept, and leave-one-out analyses were executed. Reverse-direction MR and bidirectional MR analyses evaluated potential reciprocal causation and the influence of endometriosis on immune cell composition.ResultsOur analysis identified five immune phenotypes inversely associated with endometriosis risk. These phenotypes comprise: a percentage of CD11c + HLA-DR + + monocytes, CD25 expression on CD39 + CD4 + T cells, elevated CD25 on CD45RA + CD4 + non-regulatory T cells, HLA-DR intensity on HLA-DR + CD8 bright (CD8br) T cells, and the proportion of naïve double-negative (CD4 − CD8− %DN) T cells. In contrast, eleven phenotypes were positively correlated with endometriosis risk, including: CD127 expression on T cells, the proportion of CD24 + CD27 + B cells within lymphocytes, CD25 expression on CD28 + CD4 + T cells, CD28 expression on CD39 + activated regulatory T cells (activated Tregs), the frequency of bright CD33 HLA-DR + CD14 − cells within the CD33br HLA-DR + compartment, CD45 expression on lymphocytes and natural killer (NK) cells, activation status of central memory CD8 bright (CM CD8br) T cells, CX3CR1 expression on monocytes, and the percentage of HLA-DR + NK cells within the NK cell subset. Sensitivity assessments that excluded significant heterogeneity and pleiotropy confirmed the stability of these associations, thereby reinforcing the validity of our findings.ConclusionThis study provides novel evidence of the potential causal impact of specific immune cells on the risk of developing endometriosis. These findings enhance our understanding of endometriosis pathophysiology and may inform innovative approaches for its diagnosis and management. While our findings provide novel insights, limitations such as potential horizontal pleiotropy and reliance on European ancestry data should be considered. Future research should expand to diverse populations and incorporate individual-level data to refine these findings.

Developmental trajectory of unconventional T cells of the cynomolgus macaque thymus

As nonhuman primates are immunologically the closest model to humans, a comprehensive understanding of T-cell development in these species is crucial. However, the differentiation pathways in which thymocytes participate, along with their heterogeneity, remain poorly characterized. Using single-cell RNA sequencing, we thoroughly profiled the development of various T-cell lineages in the juvenile cynomolgus monkey thymus, identifying and characterizing 12 distinct thymic cell states or types. Interestingly, we identified two unexpected cell types, an agonist-selected and a memory-like cell population. The agonist-selected cell population expressed genes associated with strong TCR signaling, such as PDCD1, CD5, NFKBID, NFATC1, BCL2L11, and NR4A1 but exhibiting significantly higher PDCD1 expression compared with cells following the conventional developmental pathway. Additionally, we identified a substantial number of memory-like cell populations characterized by high CXCR3 and EOMES expression. Notably, this population also highly expressed the effector-associated markers, GZMK, NKG7, and GNLY, as well as the innate cell-associated markers, ZBTB16, TYROBP, KLRB1, KLRC1, and NCR3. The EOMES + memory-like cell population expressed highly PDCD1, indicating the presence of an agonist-selection footprint. Our findings provide insights into the agonist-selection pathway that allows self-reactive thymocytes to survive thymic selections and differentiate into various unconventional T-cell lineages.

CXC chemokine receptor 4 - mediated immune modulation and tumor microenvironment heterogeneity in gastric cancer: Utilizing multi-omics approaches to identify potential therapeutic targets.

G-protein-coupled receptors (GPRs) are critical regulators of various biological behaviors, and their role in gastric cancer (GC) progression is gaining increasing attention. Among them, the immune regulatory mechanisms mediated by chemokine receptor 4 (CXCR4) remain insufficiently understood. This study aims to explore the immune regulatory functions of CXCR4 and the heterogeneity of the tumor microenvironment (TME) by examining GPR-related gene expression in GC. Through multi-omics approaches, including spatial transcriptomics and single-cell RNA sequencing, we investigated the oncogenic mechanisms of CXCR4, particularly its role in T cell immune exhaustion. In vitro experiments, including ELISA, PCR, CCK8 assays, cell scratch assays, and colony formation assays, were used to validate the role of CXCR4 in the migration and invasion of AGS and SNU-1 cell lines. CXCR4 silencing using siRNA further demonstrated its regulatory effects on these cellular processes. Our results revealed a strong correlation between elevated CXCR4 expression and increased exhaustion of regulatory T cells (Tregs) in the TME. Furthermore, heightened CXCR4 expression was linked to increased TME heterogeneity, driven by oxidative stress and activation of the NF-κB pathway, promoting immune evasion and tumor progression. Silencing CXCR4 significantly inhibited the invasive and proliferative abilities of AGS and SNU-1 cells, while also reducing the expression of pro-inflammatory cytokines IL-1β and interleukin-6, thus alleviating chronic inflammation and improving TME conditions. In conclusion, our comprehensive investigation highlights CXCR4 as a key mediator of TME dynamics and immune modulation in GC. Targeting CXCR4 presents a promising therapeutic strategy to slow tumor progression by reducing Tregs-mediated immune exhaustion and TME heterogeneity, positioning it as a novel therapeutic target in GC treatment.

Human microglia polarization following infection with the Lyme disease spirochete.

Infection with Borrelia burgdorferi can spread and cause central nervous system involvement, known as neuroborreliosis. Microglia phagocytose bacteria, mediate inflammation, and elicit an immune response toward the spirochete. Like other tissue macrophages, microglia can polarize into two different modulatory phenotypes, M1 and M2. We explored human microglial polarization changes upon infection with B. burgdorferi. HMC3 human microglia cell line was infected with B. burgdorferi for 24 h. Expression of polarization markers was evaluated via flow cytometry at 4 and 24 h. Secreted immunological mediators were evaluated using a multiplex ELISA system at 4, 18, and 24 h. An early decrease followed by a later increase in expression of M1 polarization marker iNOS was observed at 4 h, and 24 h, respectively. A decrease in M2 marker CX3CR1 occurred at 24 h. There were no changes in expression of M1 markers CD14, or in M2 markers CD163 and CD206. Multiplex ELISA evidenced an increase in secretion of activation markers MIP-1α, MIP- 1β, IP-10, chemotactic factor MCP-1, M1 polarization cytokine IL-8, and VEGF, at 4, 18, and 24 h. A decrease of iNOS at 4 h of infection suggests a diminished production of reactive nitrogen species that are a critical component of innate defense against infection. Increased iNOS and simultaneously decreased expression of CX3CR1 at 24 h, may suggest initiation of neuroprotective regulation of microglia recruitment to neuroinflammation. The dynamics of major inflammatory cytokines appear to be important in the microglial response to B. burgdorferi and should be further studied as these could become therapeutic targets in neuroborreliosis.

Modulation of the tumor microenvironment in non-muscle-invasive bladder cancer by OncoTherad® (MRB-CFI-1) nanoimmunotherapy: effects on tumor-associated macrophages, tumor-infiltrating lymphocytes, and monoamine oxidases.

Non-muscle-invasive bladder cancer (NMIBC) presents management challenges due to its high recurrence rate and a complex tumor microenvironment (TME). This study investigated the effects of OncoTherad® (MRB-CFI1) nanoimmunotherapy on the TME of BCG-unresponsive NMIBC, focusing on alterations in monoamine oxidases (MAO-A and MAO-B) and immune markers: CD163, FOXP3, CD8, and CX3CR1. A comparative analysis of immunoreactivities was made before and after OncoTherad® treatment and an immune score (IS) was established to evaluate the correlation between immunological changes and clinical outcomes. Forty bladder biopsies of twenty patients were divided into 2 groups (n = 20/group): 1 (pre-treatment biopsies); and 2 (post-treatment biopsies). Our results showed stable MAO-A levels but a significant (p < 0.05) decrease in MAO-B immunoreactivity after treatment, suggesting OncoTherad®'s efficacy in targeting the tumor-promoting and immunosuppressive functions of MAO-B. Significant (p < 0.05) reductions in CD163 and FOXP3 immunoreactivities were seen in post-treatment biopsies, indicating a decreased presence of M2 macrophages and Tregs. Corroborating with these results, we observed reductions in tumor histological grading, focality and size, factors that collectively enhanced recurrence-free survival (RFS) and pathological complete response (PCR). Moreover, elevated IFN-γ immunoreactivities in treated biopsies correlated with increased counts of CD8+ T cells and higher CX3CR1 expression, underscoring OncoTherad®'s enhancement of cytotoxic T cell functionality and overall antitumor immunity. The IS revealed improvements in immune responses post-treatment, with higher scores associated with better RFS and PCR outcomes. These findings validate OncoTherad®'s capability to modify the bladder cancer microenvironment favorably, promoting effective immune surveillance and response.

Expression of key immune genes in polarized porcine monocyte-derived macrophage subsets

Swine are considered one of the most relevant large animal biomedical models since they share many immunological similarities with humans. Despite that, macrophage polarization has not comprehensively investigated in pigs. In this study, porcine monocyte-derived macrophages (moMΦ) were untreated or stimulated with IFN-γ + LPS (classical activation), or by different M2 polarizing stimuli: IL-4, IL-10, TGF-β, or dexamethasone. Expression of key cytokine genes (IL1B2, IL33, IL19, IL22, IL26, CCL17, CCL24, IFNA, IFNB) in macrophage subsets were investigated over time. Expression of the genes encoding the two main enzymes of the arginine pathway (ARG1, NOS2), and molecules related to alternative macrophage polarization in human and mice (MMP9, MRC1, FIZZ1, VEGFA) were also assessed. Stimulation with IFN-γ + LPS triggered up-regulation of IL1B2, IFNB, NOS2, whereas IL-4 triggered upregulation of CCL17, CCL24, CXCR2, and ARG1 expression. IL19 and IL22 expression was enhanced by stimulation with IFN-γ + LPS or TGF-β, but not IL-4, IL-10, or dexamethasone. Our data highlighted some peculiarities in swine, such as induced expression of IL33 after stimulation with IFN-γ + LPS, and no up-regulation of FIZZ1, VEGFA or MMP9 after exposure to any of the M2 polarizing stimuli. A better understanding of porcine macrophage polarization could benefit translational studies using this large animal model.

MiR-147-3p in pathogenic CD4 T cells controls chemokine receptor expression for the development of experimental autoimmune diseases

Incomplete Freund's adjuvant (IFA) has long been used to trigger autoimmune diseases in animal models, such as experimental autoimmune encephalitis and collagen-induced arthritis. However, the molecular mechanisms that control CD4 T cell effector functions and lead to the development of autoimmune diseases are not well understood. A self-antigen and heat-killed Mycobacterium tuberculosis emulsified in IFA augmented the activation of CD4 T cells, leading to the differentiation of pathogenic CD4 T cells in the draining lymph nodes. In contrast, IFA emulsification did not elicit Foxp3+ regulatory T cell expansion. We found that pathogenic Th1 cells expressed miR-147-3p, which targets multiple genes to affect T cell function. Finally, miR-147-3p expressed in CXCR6+SLAMF6– Th1 cells was required for the onset of neurological symptoms through the control of CXCR3 expression. Our findings demonstrate that miR-147-3p expressed in pathogenic CD4 T cells regulates the migratory potential in peripheral tissues and impacts the development of autoimmune diseases.