Chemokines CXCL10 Research Articles

Assessment of threat of concurrent SARS-CoV-2 and DENV infection in the COVID-19 pandemic in Brazil in 2020: diagnostic and immunological findings

IntroductionThe first peak of COVID-19 in Brazil was between April and May 2020, at a time of the year when outbreaks of other tropical diseases, such as dengue, would be expected. COVID-19 and dengue have similar pathogenesis. In general, both may lead to mild symptoms but may also cause severe and even fatal symptoms, especially in patients with comorbidities and probably in cases of overlapping infections. The general objective of this study was to assess whether, during the 2020 pandemic, there were cases of concomitant infection between SARS-CoV-2 and DENV.MethodsFor this, we evaluated the specificity and sensitivity of commercial serological anti-SARS-CoV-2 kits using plasma samples from patients with dengue and healthy donors recruited before COVID-19. In the case of confirmed cases of COVID-19/dengue, we evaluated the clinical evolution of these coinfected patients, compared with mono-infected patients; and quantified chemokines CCL2 and CXCL8 by ELISA in COVID-19 patients in order to correlate them with COVID-19/dengue severity and cases.Results and DiscussionOur results showed that commercial IgA and IgG anti-SARS-CoV-2 kits presented high sensitivity and specificity. This allowed us to see a low rate of co-detection or coinfection between SARS-CoV-2 and DENV in Rio de Janeiro. Among the 57 COVID-19 patients, anti-DENV IgM was detected in five (8.8%). COVID-19/dengue coinfected patients showed no clinical worsening of COVID-19 and cases in which COVID-19 patients had previous exposure to DENV did not influence the clinical severity of COVID-19. Lastly, CCL2 and CXCL8 appeared to be good markers of COVID-19 severity and did not show increased levels in COVID-19/dengue cases.

What is the impact of e-cigarettes on periodontal stem cells as revealed by transcriptomic analyses?

The research used an in vitro cell exposure model and multi-omics integration of transcriptome and epigenome profiling to compare the molecular effects of e-cigarettes and tobacco smoke on dental stem cells. The study aimed to compare the effects of e-cigarette and tobacco smoke on periodontal stem cells using a multi-omics approach to understand gene regulation. This research studied primary human gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) obtained from healthy donors. The cells were subjected to tobacco smoke, e-cigarette aerosol (both tobacco and menthol flavors), e-cigarette liquid (both tobacco and menthol flavors), or untreated conditions using an in vitro exposure system. RNA sequencing and bioinformatics analysis were used to profile the transcriptome and identify differential gene expression. Additionally, chromatin immunoprecipitation sequencing (ChIP-seq) was used to conduct genome-wide histone modification mapping for H3K27me3. Transcriptome profiling was combined with histone modification characterization to understand gene regulatory mechanisms. The study compared the effects of smoke versus e-cigarette, aerosol versus liquid exposure, and tobacco versus menthol flavor on gene expression and epigenetic landscapes in the two oral stem cell populations. The use of tobacco smoke caused damage to the DNA and nucleus in GMSCs, as well as mitochondrial dysfunction in PDLSCs. Regarding e-cigarettes, the aerosol and liquid affected non-coding RNA expression differently. The chemokine CXCL2 was found to be downregulated by aerosol but upregulated by liquid in GMSCs. An integrative analysis revealed that the upregulation of CXCL2 caused by e-liquid involved reduced H3K27me3 and activation of distal enhancers. On the other hand, aerosol exposure maintained H3K27me3 levels, while direct e-liquid exposure resulted in genome-wide reductions in H3K27me3, particularly in enhancer regions. Overall, the specific delivery methods and components of e-cigarettes caused unique changes in the transcriptome and epigenome of oral stem cells. E-cigarettes affect oral stem cells differently than tobacco smoke. Their aerosol and liquid have varying impacts on gene expression and regulatory landscapes in oral cells. Multi-omics approaches are important to understanding the molecular changes caused by e-cigarette components. This can help with toxicological assessments and determine their impact on periodontal health. Transcriptome and epigenome profiling are powerful tools to examine the unique molecular mechanisms involved in cellular responses to e-cigarettes.

CXCL1 promotes immune escape in colorectal cancer by autophagy-mediated MHC-I degradation

BackgroundImmunotherapy is now seen as a potential remedy for colorectal cancer (CRC). Chemokines play a crucial role in tumors, including CRC, which contains CXCL1. We attempted to study how CXCL1 impacts immune escape in CRC. MethodsBioinformatics analysis was used to examine CXCL1 level in CRC. qRT-PCR was used to assess CXCL1 and MHC-I (HLA-A, B, C) levels. Cell Counting Kit-8 (CCK-8) was used to measure cell viability. Cytotoxicity assay kit was utilized to assay CD8+ T cell cytotoxicity against CRC. Flow cytometry tested proliferation and apoptosis of CD8+ T cells. Chemotaxis assay evaluated chemotaxis of CD8+ T cells towards CRC. Immunofluorescence examined expression of autophagy marker LC3 and localization of NBR1/MHC-I. Western blot analysis measured protein levels of chemokines CXCL9 and CXCL10, autophagy-related proteins LC3-I and LC3-II, and MHC-I (HLA-A, B, C). ResultsBioinformatics analysis and qRT-PCR presented that CXCL1 was upregulated in CRC. Cell experiments demonstrated that CXCL1 overexpression promoted immune escape in CRC. Rescue experiments revealed that the autophagy inducer Rapa could attenuate the inhibitory effect of CXCL1 low expression on immune escape in CRC. Further studies showed that CXCL1 promoted immune escape in CRC by autophagy-mediated MHC-I degradation. ConclusionCXCL1 promoted immune escape in CRC by autophagy-mediated MHC-I degradation, suggesting that CXCL1 may be a possible immunotherapeutic target for CRC.

Automated Urinary Chemokine Assays for Noninvasive Detection of Kidney Transplant Rejection: A Prospective Cohort Study

Prior studies have demonstrated the diagnostic potential of urinary chemokines C-X-C motif ligand 9 (CXCL9) and CXCL10 for kidney transplant rejection. However, their benefit in addition to clinical information has not been demonstrated. We evaluated the diagnostic performance for detecting acute rejection of urinary CXCL9 and CXCL10 when integrated with clinical information. Single-center prospective cohort study. We analyzed 1,559 biopsy-paired urinary samples from 622 kidney transplants performed between April 2013 and July 2019 at a single transplant center in Belgium. External validation was performed in 986 biopsy-paired urinary samples. We quantified urinary CXCL9 (uCXCL9) and CXCL10 (uCXCL10) using an automated immunoassay platform and normalized the values to urinary creatinine. Urinary chemokines were incorporated into a multivariable model with routine clinical markers (estimated glomerular filtration rate, donor-specific antibodies, and polyoma viremia) (integrated model). This model was then compared with the tissue diagnosis according to the Banff classification for acute rejection. Acute rejection detected on kidney biopsy using the Banff classification. Chemokines integrated with routine clinical markers had high diagnostic value for detection of acute rejection (n=150) (receiver operating characteristic area under the curve 81.3% [95% CI, 77.6-85.0]). The integrated model would help avoid 59 protocol biopsies per 100 patients when the risk for rejection is predicted to be below 10%. The performance of the integrated model was similar in the external validation cohort. The cross-sectional nature obviates investigating the evolution over time and prediction of future rejection. The use of an integrated model of urinary chemokines and clinical markers for noninvasive monitoring of rejection could enable a reduction in the number of biopsies. Urinary chemokines may be useful noninvasive biomarkers whose use should be further studied in prospective randomized trials to clarify their role in guiding clinical care and the use of biopsies to detect rejection after kidney transplantation. Urinary chemokines CXCL9 and CXCL10 have been suggested to be good noninvasive biomarkers of kidney transplant rejection. However, defining a context of use and integration with clinical information is necessary before clinical implementation can begin. In this study, we demonstrated that urinary chemokines CXCL9 and CXCL10, together with clinical information, have substantial diagnostic accuracy for the detection of acute kidney transplant rejection. Application of urinary chemokines together with clinical information may guide biopsy practices following kidney transplantation and potentially reduce the need for kidney transplant biopsies.

PARP Inhibitor Plus Radiotherapy Reshapes IDH1 Mutation Tumor Immune Suppression Microenvironment Potentiating the Efficiency of Immune Checkpoint Inhibitor

Isocitrate dehydrogenase 1 (IDH1) mutations confer gain-of-function activity by converting α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG). IDH mutant tumors have fewer tumor-infiltrating CD8+ T cells and reduced PD-L1 expression compared with their wild type (WT) counterparts. In addition, 2-HG can directly inhibit the killing and proliferative functions of CD8+ T lymphocytes, and suggesting that 2-HG promotes an immunosuppressive TME. Several studies have shown that 2-HG can inhibit homologous recombination (HR) and weaken the DNA damage response (DDR), making them more sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors and radiotherapy (RT). At the same time, RT and PARP inhibition (PARPi) have been considered to be a new direction to stimulate antitumor immunity. Therefore, our study intends to use RT + PARPi to reverse the immunosuppressive microenvironment caused by IDH1 mutations, thereby promoting the therapeutic effect of immune checkpoint inhibitors. We compared the immune responses of clinical tissue samples and TCGA data from either IDH1mut or IDH1WT low-grade gliomas. We then established IDH1mut-overexpressing MC38 and GL261 cell lines to determine the antitumor effect of RT + PARPi. Apoptosis and immunogenic death markers were detected by flow cytometry, western blot (WB) and ELISA in these cell lines. Tumor growth and mouse survival curves were observed in both an MC38 subcutaneous and GL261 orthotopic tumor model. Changes in the composition of the immune microenvironment were assessed using flow cytometry. The mechanisms underpinning these compositional shifts were then further interrogated using various techniques, including WB, immunofluorescence, qRT-PCR, CRISPR/Cas9, and CD8+ T cell migration experiments. We observed that CD8+ T cell infiltration and expression of the chemokines CXCL10 and CCL5 of CD8+ T cells in IDH1mut tumors were significantly downregulated by immunohistochemistry and TCGA analysis. Gene enrichment analysis using the TCGA database found that IDH1 mutations downregulated interferon (IFN)-related signaling pathways. RT + PARPi induces more DNA damage and actives the CGAS-STING pathway compared with monotherapy, leading to more expression of IFN-β, CXCL10 and CCL5 at mRNA and protein level. In the MC38 subcutaneous tumor model, we found that RT + PARPi increased the infiltration of CD8+ T cells while enhancing the killing function of CD8+ T cells. We observed these same effects in the GL261 orthoma model, as well as increased proliferation function of CD8+ T cells. In addition, RT + PARPi increased the expression of PD-L1 and enhanced the therapeutic effect of immune checkpoint inhibitors. RT + PARPi reshapes the IDH1mut tumor immune suppression microenvironment, thereby potentiating the antitumor effect and efficiency of immune checkpoint inhibitor.

Doxorubicin Enhances the Abscopal Effect Depending on Tumor Cell Mitochondrial DNA and cGAS/STING

Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, this effect is still clinically rare and improvements are highly desirable. We investigated whether triple combination with a low dose of clinically approved liposomal doxorubicin (Doxil) could augment abscopal responses compared with RT+ICB, Doxil+ICB, or RT+Doxil. We used Doxil in combination with RT and αPD1 in two tumor models (B16-CD133 melanoma and MC38 colon carcinoma) with mice bearing two tumors, only one of which was irradiated. Triple therapy with RT, αPD1, and single low-dose Doxil strongly enhanced the RT-induced abscopal effect compared to all double and single treatments in both tumor models (p < 0.05, n = 5-10 mice/group). Complete cures of non-irradiated tumors were mainly observed in triple-treated mice. Triple therapy induced more cross-presenting dendritic cells (DCs) and tumor-specific CD8 T cells than RT/αPD1 and Doxil/αPD1 (p < 0.05, n = 5 mice/group), particularly in non-irradiated tumors. CD8 T cell depletion or implanting STING-deficient tumor cells abolished the abscopal effect. By using inhibitors and knockout cells, we show that doxorubicin/Doxil-induced IFNβ1 markedly depended on the cGAS/STING pathway (p < 0.05) which drives antitumor CD8 T cell responses through cross-presenting DCs. In mitochondrial DNA (mtDNA)-depleted tumor cells, doxorubicin/Doxil induced less IFNβ1 (p < 0.05), the related T cell-recruiting chemokine CXCL10 (p < 0.0001), and ATP (p < 0.0001); coincubation with mtDNA-depleted tumor cells strongly reduced IFNβ1 (p < 0.01) secretion by DCs. Implantation of mtDNA-depleted tumor cells, particularly at the non-irradiated site, substantially diminished the Doxil-enhanced abscopal effect and tumor infiltration by tumor-specific CD8 T cells (p < 0.05). Single low-dose Doxil can substantially enhance the RT-induced abscopal effect, with a strong increase in cross-presenting DCs and CD8 tumor-specific T cells particularly in abscopal tumors compared with RT/αPD1 and Doxil/αPD1. The mtDNA/cGAS/STING/IFN-I axis is important for the immunogenic doxorubicin effects. Our findings may be helpful for the planning of clinical radiochemoimmunotherapy trials in (oligo)metastatic patients.

The Effect of PARP Inhibitor Radiosensitization on the mRNA Translational Regulation of T Cell Chemokines

Immunotherapy has modestly improved survival for small cell lung cancer (SCLC) patients. Low response rate and rapid disease progression remain an intractable challenge. One of the factors that contribute to immunotherapy resistance is the lack of cytotoxic T cell infiltration. The expression of chemoattractant cytokines, like CCL5 and CXCL10, are essential for T cell infiltration. The control of chemokine expression is not fully understood, but both transcriptional and translational control pathways could play a major role. Previous studies have shown a correlation between DNA damage and chemokine expression and that PARP inhibitors (PARPi) are radiosensitizers for SCLC that increases DNA damage. The objectives of this study were to define this potential PARPi immunogenic radiosensitizing relationship. We identified doses of olaparib+ radiation treatment (RT) that conferred radiosensitization in SCLC cell-lines by cell viability and/or clonogenic assays. Olaparib+RT induced CCL5 and CXCL10 mRNA expression was measured by qPCR across SCLC cell-lines. Protein level of chemokines was assessed by immunoblotting. SBC5 cells were treated with olaparib+RT and submitted for RNA sequencing analysis. Genes with adjusted p value<0.05 were considered significant. Protein level changes and target gene knock-out (KO) were confirmed by immunoblotting. Chemokine CXCL10 mRNA and protein level in wildtype (WT) and KO cells were measured by qPCR and western blot, respectively. A mRNA decay assay and dual-luciferase reporter assay was used to identify the region of CXCL10 mRNA that confers mRNA stability control. In vivo anti-tumor efficacy and tumor T cell infiltration studies were done in B6129F mice bearing KP1 tumors. And the T cell infiltration was measured by immune profiling. In vitro, olaparib+RT significantly increased CXCL10 mRNA in all four SCLC subtype cell-lines in comparison to vehicle control. Consistently, the increase of CXCL10 protein levels (3-fold) was observed in SBC5 cells. By RNA-Seq, a top-ranking translational repressor was EIF4E2 (4EHP) mRNA. The downregulation of EIF4E2 protein by olaparib+RT was validated in four SCLC subtypes by western blot. EIF4E2 KO in HEK293 and SBC5 cells increased CXCL10 mRNA and protein level. By mRNA decay assay and western blot, the absence of EIF4E2 stabilized CXCL10 mRNA and increased CXCL10 protein levels. The dual-luciferase assay demonstrated EIF4E2 destabilizes CXCL10 mRNA via the 3'UTR of CXCL10. In vivo, immune profiling showed olaparib+RT significantly increased the total T cell and CD8+ T cell infiltration. Finally, anti-PD-L1 inhibition potentiated olaparib + IR to improve tumor control in KP1 allograft. Our study demonstrated olaparib + RT increases CXCL10 protein levels through downregulating EIF4E2 to subsequently increase T cell infiltration. Olaparib + RT enhanced anti-PD-L1 immunotherapy efficacy and has therapeutic potential as an immunogenic radiosensitizer.

Role of the transcription factor Fli-1 on the CXCL10/CXCR3 Axis.

The transcription factor Fli-1, a member of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNFα stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment.

Afferent neurons of the kidney with impaired firing pattern in inflammation - role of sodium currents?

Peripheral neurons with renal afferents exhibit a predominantly tonic firing pattern of higher frequency that is reduced to low frequencies (phasic firing pattern) in renal inflammation. We wanted to test the hypothesis that the reduction in firing activity during inflammation is due to high-activity tonic neurons switching from higher to low frequencies depending on altered sodium currents. We identified and cultivated afferent sensory neurons with renal projections from the dorsal root ganglia (Th11-L2). Cultivated neurons were incubated with the chemokine CXCL1 (1,5nmol/ml) for 12h. We characterized neurons as "tonic," i.e., sustained action potential (AP) firing, or "phasic," i.e., < 5 APs upon stimulation in the current clamp. Their membrane currents were investigated in a voltage clamp. Data analyzed: renal vs. non-renal and tonic vs. phasic neurons. Renal afferent neurons exposed to CXCL1 showed a decrease in tonic firing pattern (CXCL1: 35,6% vs. control: 57%, P < 0.05). Na+ and K+ currents were not different between control renal and non-renal DRG neurons. Phasic neurons exhibited higher Na+ and K+ currents than tonic resulting in shorter APs (3.7 ± 0.3 vs. 6.1 ± 0.6ms, P < 0.01). In neurons incubated with CXCL1, Na+ and K+ peak current density increased in phasic (Na+: - 969 ± 47 vs. - 758 ± 47 nA/pF, P < 0.01; K+: 707 ± 22 vs. 558 ± 31 nA/pF, P < 0.01), but were unchanged in tonic neurons. Phasic neurons exposed to CXCL1 showed a broader range of Na+ currents ([- 365- - 1429 nA] vs. [- 412- - 4273 nA]; P < 0.05) similar to tonic neurons. After CXCL1 exposure, significant changes in phasic neurons were observed in sodium activation/inactivation as well as a wider distribution of Na+ currents characteristic of tonic neurons. These findings indicate a subgroup of tonic neurons besides mere tonic or phasic neurons exists able to exhibit a phasic activity pattern under pathological conditions.

Decidualized human decidual stromal cells inhibit chemotaxis of activated T cells: a potential mechanism of maternal-fetal immune tolerance.

Numerous lines of evidence confirm that decidual stromal cells (DSCs) play a key role in maternal-fetal immune tolerance. Under the influence of progesterone and other hormones, the DSCs go through a process of differentiation (decidualization) during normal pregnancy. In mice, DSCs inhibit the expression of chemokines that attract abortigenic Th1 and Tc cells to the decidua. We have studied this phenomenon in humans. We established human DSC lines and decidualized these cells in vitro with progesterone and cAMP. We determined the expression of the chemokines CXCL9, CXCL10 and CXCL11, whose receptor CXCR3 is expressed by Th1 and Tc cells, in undifferentiated DSCs and decidualized DSCs by qRT-PCR. Activated CD3+CXCR3+ cells, including CD4+ Th1 cells and CD8+ Tc cells, were induced in vitro. The migration capacity of these activated lymphocytes was investigated in Transwell chambers with conditioned media from undifferentiated and decidualized DSCs. We demonstrated that CXCL9 was not expressed by DSCs, whereas the expression of CXCL10 and CXCL11 was inhibited in decidualized cells. Conditioned media from decidualized cells significantly inhibited the migration of Th1 and Tc cells. We found that decidualized cells secrete factors of MW less than 6000-8000 Da, which actively inhibit the chemotaxis of these lymphocytes. These results confirm in humans that decidualization of DSCs inhibits the expression by these cells of chemokines that attract Th1 and Tc cells and induces the secretion by DSCs of factors that inhibit the chemotaxis of these lymphocytes, thus preventing the arrival of abortigenic T cells in the decidua.

Sulfation Effects of Chondroitin Sulfate to Bind a Chemokine in Aqueous Medium: Conformational Heterogeneity and Dynamics from Molecular Simulation.

The sulfation patterns and degree of sulfation of chondroitin sulfate (CS), an important class of glycosaminoglycans (GAG), and their interactions with chemokines are accountable for various diseases. To realize the underlying mechanism of such complex biological phenomena at a molecular level and their application in rational drug design, a study on conformations and dynamics of CSs is necessary. To explore this, in this study, we performed a series of atomistic molecular dynamics (MD) simulations with different sulfated variants of octadecasaccharide CS, like CS-C, CS-E, and CS-T, in their free forms and when bound to the protein chemokine CXCL8 dimer in an aqueous medium. The calculated binding free energy of CSs with the CXCL8 dimer is favorable, and the degree of sulfation favors the complexation process further with prominent hydrophobic and hydrogen-bonded interactions. We find that the recognition is associated with the configurational entropy loss of the CS molecules as calculated from the Gaussian mixture approach, which supports that the degree of sulfation regulates the process. Cluster analysis through the k-means algorithm and end-to-end distance measurement revealed that although the free CS molecules adopted linear conformations, the nonlinear conformations during binding with protein were noted. Adaptation of nonlinear forms in the bound forms is noteworthy for the less-sulfated CS-C and CS-E. Apart from favorable 4C1 conformations, the occasional appearance of skew-boat forms from the free-energy map of ring pucker for the GlcUA unit was observed, which remains unaffected by the sulfation. We find that during recognition, the average relaxation time of intra-CS and inter-CS-CXCL8 hydrogen bonds (HBs) is about a magnitude lesser than that of CS-water HBs, most prominent on the involvement of higher sulfated CS-T analogues. The translational motion of surrounded water molecules in CSs exhibited sublinear diffusion, and the degree of sublinearity increases around the heavily sulfated molecules due to the hindrance created by them as well as the presence of the chemokine and exhibited markedly slow heterogeneous diffusion.

Pansclerotic morphea is characterized by IFN-γ responses priming dendritic cell fibroblast crosstalk to promote fibrosis.

Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and to identify therapeutically targetable pathways by performing single-cell and spatial RNA-Seq on 7 healthy controls and on lesional and nonlesional skin biopsies of a patient with PSM 12 months apart. We then validated our findings using immunostaining and in vitro approaches. Fibrotic skin was characterized by prominent type II IFN response, accompanied by infiltrating myeloid cells, B cells, and T cells, which were the main IFN-γ source. We identified unique CXCL9+ fibroblasts enriched in PSM, characterized by increased chemokine expression, including CXCL9, CXCL10, and CCL2. CXCL9+ fibroblasts were related to profibrotic COL8A1+ myofibroblasts, which had enriched TGF-β response. In vitro, TGF-β and IFN-γ synergistically increased CXCL9 and CXCL10 expression, contributing to the perpetuation of IFN-γ responses. Furthermore, cell-to-cell interaction analyses revealed cDC2B DCs as a key communication hub between CXCL9+ fibroblasts and COL8A1+ myofibroblasts. These results define PSM as an inflammation-driven condition centered on type II IFN responses. This work identified key pathogenic circuits between T cells, cDC2Bs, and myofibroblasts, and it suggests that JAK1/2 inhibition is a potential therapeutic option in PSM.

BSBM-20 INVESTIGATING THE ROLE OF OXIDATIVE PHOSPHORYLATION (OXPHOS) IN CD4+ T CELL ACTIVATION AND RESPONSE TO ANTI-PD1/ANTI-LAG3 IMMUNOTHERAPY IN MELANOMA BRAIN METASTASIS (MBM)

Abstract Brain metastasis is a frequent and deadly complication of metastatic melanoma. While extracranial metastases (ECMs) are often treatable, MBMs remain a critical treatment (tx) challenge to overcome. We previously showed that MBMs are characterized by increased utilization of OXPHOS compared to ECMs, and OXPHOS inhibition increased expression of immune activation transcriptional signatures in MBMs. Thus, we tested the role of tumor OXPHOS on the immune microenvironment and checkpoint immunotherapy response in a syngeneic mouse model with concurrent intracranial (ICr) and subcutaneous (SQ) tumors. Selective OXPHOS inhibition was achieved via CRISPR-KO of Ndufs4 (mitochondrial complex I) in B16-F10 cells and confirmed via Seahorse Analyzer MitoStress Test. Multiplex cytokine analysis of culture supernatant revealed that NDUFS4 KO increased anti-tumor cytokines IFNγ (p=0.016) and IL-2 (p=0.023) and decreased pro-tumor chemokine CXCL2 (p&amp;lt;0.001). Next, we implanted concurrent ICr and SQ B16-F10 tumors in C57BL/6J mice. Flow cytometry demonstrated decreased CD4+ and CD8+ T cell infiltration of ICr versus SQ tumors in both B16-F10 and NDUFS4 KO tumors (all p&amp;lt;0.001). NDUFS4 KO increased total CD4+ T cells (p=0.041) and activated CD4+ T cells (p&amp;lt;0.001) in ICr tumors. Finally, we tested the efficacy of tx with combination anti-PD1/anti-LAG3 (200μg/100μg intraperitoneal 3x/week) versus isotype controls (Ctrl) in these mice. Compared to Wildtype (WT) tumors with Ctrl tx, we observed improved overall survival (OS) in mice with NDUFS4 KO tumors + Ctrl (HR 0.117; p=0.015), WT tumors + anti-PD1/anti-LAG3 (HR 0.071; p=0.005), and NDUFS4 KO tumors + anti-PD1/anti-LAG3 (HR 0.049; p=0.002). For anti-PD1/anti-LAG3 tx, there was a trend for improved OS with NDUFS4 KO versus WT tumors (HR 0.317; p=0.130). Together, these data implicate tumor OXPHOS in CD4+ T cell immunosuppression and a potential target to enhance response to checkpoint immunotherapy in MBMs.

Adding liposomal doxorubicin enhances the abscopal effect induced by radiation/αPD1 therapy depending on tumor cell mitochondrial DNA and cGAS/STING

BackgroundLocalized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade. However, this effect is still clinically rare and improvements...

Bradykinin B1 Receptor Affects Tumor-Associated Macrophage Activity and Glioblastoma Progression.

Bradykinin is a small active peptide and is considered an inflammatory mediator in several pathological conditions. Bradykinin exerts its effects by coupling to its receptors, including bradykinin B1 (B1R) and bradykinin B2. B1R has been implicated in the development of various cancers. Our previous study reported that B1R promoted glioblastoma (GBM) development by supporting the migration and invasion of GBM cells. However, the mechanisms underlying the effects of B1R on tumor-associated macrophages (TAMs) and GBM progression remain unknown. Accordingly, to explore the regulatory effects of B1R overexpression (OE) in GBM on tumor-associated immune cells and tumor progression, we constructed a B1R wild-type plasmid and developed a B1R OE model. The results reveal that B1R OE in GBM promoted the expression of ICAM-1 and VCAM-1-cell adhesion molecules-in GBM. Moreover, B1R OE enhanced GBM cell migration ability and monocyte attachment. B1R also regulated the production of the protumorigenic cytokines and chemokines IL-6, IL-8, CXCL11, and CCL5 in GBM, which contributed to tumor progression. We additionally noted that B1R OE in GBM increased the expression of CD68 in TAMs. Furthermore, B1R OE reduced the level of reactive oxygen species in GBM cells by upregulating heme oxygenase-1, an endogenous antioxidant protein, thereby protecting GBM cells from oxidative stress. Notably, B1R OE upregulated the expression of programmed death-ligand 1 in both GBM cells and macrophages, thus providing resistance against T-cell response. B1R OE in GBM also promoted tumor growth and reduced survival rates in an intracranial xenograft mouse model. These results indicate that B1R expression in GBM promotes TAM activity and modulates GBM progression. Therefore, B1R could be an effective target for therapeutic methods in GBM.

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury

Liver sinusoidal endothelial cells (LSECs) rapidly clear lipopolysaccharide (LPS) from the bloodstream and establish intimate contact with immune cells. However, their role in regulating liver inflammation remains poorly understood. We show that LSECs modify their chemokine expression profile driven by LPS or interferon-γ (IFN-γ), resulting in the production of the myeloid- or lymphoid-attracting chemokines CCL2 and CXCL10, respectively, which accumulate in the serum of LPS-challenged animals. Natural killer (NK) cell exposure to LSECs invitro primes NK cells for higher production of IFN-γ in response to interleukin-12 (IL-12) and IL-18. In livers of LPS-injected mice, NK cells are the major producers of this cytokine. In turn, LSECs require exposure to IFN-γ for CXCL10 expression, and endothelial-specific Cxcl10 gene deletion curtails NK cell accumulation in the inflamed livers. Thus, LSECs respond to both LPS and immune-derived signals and fuel a positive feedback loop of immune cell attraction and activation in the inflamed liver tissue.

Dynamics of reactive astrocytes fosters tissue regeneration after cuprizone-induced demyelination.

Demyelination in the central nervous system (CNS) is a hallmark of many neurodegenerative diseases such as multiple sclerosis (MS) and others. Here, we studied astrocytes during de- and remyelination in the cuprizone mouse model. To this end, we exploited the ribosomal tagging (RiboTag) technology that is based on Cre-mediated cell type-selective HA-tagging of ribosomes. Analyses were performed in the corpus callosum of GFAP-Cre+/- Rpl22HA/wt mice 5 weeks after cuprizone feeding, at the peak of demyelination, and 0.5 and 2 weeks after cuprizone withdrawal, when remyelination and tissue repair is initiated. After 5 weeks of cuprizone feeding, reactive astrocytes showed inflammatory signatures with enhanced expression of genes that modulate leukocyte migration (Tlr2, Cd86, Parp14) and they produced the chemokine CXCL10, as verified by histology. Furthermore, demyelination-induced reactive astrocytes expressed numerous ligands including Cx3cl1, Csf1, Il34, and Gas6 that act on homeostatic as well as activated microglia and thus potentially mediate activation and recruitment of microglia and enhancement of their phagocytotic activity. During early remyelination, HA-tagged cells displayed reduced inflammatory response signatures, as indicated by shutdown of CXCL10 production, and enhanced expression of osteopontin (SPP1) as well as of factors that are relevant for tissue remodeling (Timp1), regeneration and axonal repair. During late remyelination, the signatures shifted towards resolving inflammation by active suppression of lymphocyte activation and differentiation and support of glia cell differentiation. In conclusion, we detected highly dynamic astroglial transcriptomic signatures in the cuprizone model, which reflects excessive communication among glia cells and highlights different astrocyte functions during neurodegeneration and regeneration.

Therapy of autoimmune inflammation in sporadic amyotrophic lateral sclerosis: Dimethyl fumarate and H-151 downregulate inflammatory cytokines in the cGAS-STING pathway.

In sporadic amyotrophic lateral sclerosis (sALS), IL-17A- and granzyme-positive cytotoxic T lymphocytes (CTL), IL-17A-positive mast cells, and inflammatory macrophages invade the brain and spinal cord. In some patients, the disease starts following a trauma or a severe infection. We examined cytokines and cytokine regulators over the disease course and found that, since the early stages, peripheral blood mononuclear cells (PBMC) exhibit increased expression of inflammatory cytokines IL-12A, IFN-γ, and TNF-α, as well as granzymes and the transcription factors STAT3 and STAT4. In later stages, PBMCs upregulated the autoimmunity-associated cytokines IL-23A and IL-17B, and the chemokines CXCL9 and CXCL10, which attract CTL and monocytes into the central nervous system. The inflammation is fueled by the downregulation of IL-10, TGFβ, and the inhibitory T-cell co-receptors CTLA4, LAG3, and PD-1, and, in vitro, by stimulation with the ligand PD-L1. We investigated in two sALS patients the regulation of the macrophage transcriptome by dimethyl fumarate (DMF), a drug approved against multiple sclerosis and psoriasis, and the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway inhibitor H-151. Both DMF and H-151 downregulated the expression of granzymes and the pro-inflammatory cytokines IL-1β, IL-6, IL-15, IL-23A, and IFN-γ, and induced a pro-resolution macrophage phenotype. The eicosanoid epoxyeicosatrienoic acids (EET) from arachidonic acid was anti-inflammatory in synergy with DMF. H-151 and DMF are thus candidate drugs targeting the inflammation and autoimmunity in sALS via modulation of the NFκB and cGAS/STING pathways.

Structural basis of CXC chemokine receptor 1 ligand binding and activation

Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.

Implantation microenvironment in chronic endometritis

Objective. To study the immune patterns of the endometrium in women with infertility of uterine genesis in the phase of "implantation window". Materials and methods. Forty-two infertile women of reproductive age were prospectively examined. At the first stage, the contingent of women was divided into groups in accordance with the causes of infertility diagnosed before the current treatment: with chronic endometritis (CE) (n = 10); with tubal peritoneal infertility (TPI) (n = 32). A comprehensive examination of women included sonography of pelvic organs, hysteroscopy, examination of endometrial material in the period of the "implantation window" (morphological, immunohistochemical, real time (RT) PCR study,). The resulting for distinguishing molecular phenotypes of chronic inflammation (n = 30) and "normal" endometrium (n = 12) were the data of immunohistochemical studies of the immune profile (in the glandular epithelium and stromal cells (TNF-, IL-10, NRF2, GM-CSF and CXCL16), in the glandular epithelium BCA1, in the stroma TGF-) compared with the indicators of healthy fertile women (control group, n = 10). CE was verified based on pathomorphological and immunohistochemical studies (CD 138+). Results. Structural features of the endometrium in women with CE in the phase of the "implantation window" corresponded to the average stage of secretion in 36.4 %; variants of "out-of-phase" were identified in 63.6 % (late stage of the proliferation phase (16.7 %), dissociated development (13.3 %), early stage of the secretion phase (43.3 %)). Molecular characteristics of the immunologically tolerant endometrium, favorable for implantation, are determined by the balanced production of pro-and anti-inflammatory cytokines, growth factors and chemokines. Aggressive "microenvironment" in the endometrium of women with the phenotype of chronic inflammation was determined by the dominant of proinflammatory cytokines of the Th1/Th17 profile (an increase in comparison with the control of the expression level of TNF- and GM-CSF by 1.1 times and 1.2 times, in the glandular epithelium of chemokines CXCL16 and BCA1 by 1.2 times, CXCL16 in the stroma in 1.2 times) on the background of reduced production of protective factors (TGF- (1 point), IL-10 (in the glandular epithelium by 2 times, in the stroma by 1.8 times), NRF2. Conclusions. Different molecular characteristics of the endometrium in women with TPI and CE determine the heterogeneity of potencies for blastocyst implantation.