CXCL10 Production Research Articles

Activation of Janus kinase 2 contributes to the autoimmune pathology in the salivary glands of patients with Sjögren's syndrome

AbstractAimPrimary Sjögren's syndrome (pSS) is a chronic autoimmune disease that affects exocrine glands. CXCL10 production from salivary gland ductal cells via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been suggested to be involved in glandular inflammation in pSS. We aimed to investigate JAK1, JAK2, phosphorylated JAK1, and phosphorylated JAK2 expression in labial salivary gland (LSG) tissues from patients with pSS to evaluate the potential of JAK inhibitors as therapeutic agents for pSS.MethodsImmunohistochemical analysis was performed using LSG tissues of patients with pSS (n = 10), non‐SS patients (n = 5), and healthy controls (n = 5). The LSG sections were scored to determine the expression levels of JAK1, JAK2, phosphorylated JAK1, and phosphorylated JAK2 in the ductal and acinar epithelium.ResultsIn acinar epithelial cells of LSG tissues, JAK1, JAK2, and phosphorylated JAK1 expression was significantly lower in patients with pSS than in the controls. Significantly high expression of phosphorylated JAK1 and phosphorylated JAK2 was observed in the ductal epithelial cells of patients with pSS. However, there was no significant association between phosphorylated JAK1 or JAK2 expression levels and inflammation degree. Furthermore, immunofluorescence analysis revealed JAK2 phosphorylation in many CD3+ T cells infiltrating the LSG tissues.ConclusionsThe results suggest JAK2 activation in both ductal epithelial cells and infiltrating CD3+ T cells in LSG tissues of patients with pSS. JAK inhibitors may be effective therapeutic agents for pSS by regulating both chemokine production from salivary gland cells and effector T cell activation.

The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation

Acute phase proteins involved in chronic inflammatory diseases have not been systematically analyzed. Here, global proteome profiling of serum and urine revealed that orosomucoid-2 (ORM2), an acute phase reactant, was differentially expressed in rheumatoid arthritis (RA) patients and showed the highest fold change. Therefore, we questioned the extent to which ORM2, which is produced mainly in the liver, actively participates in rheumatoid inflammation. Surprisingly, ORM2 expression was upregulated in the synovial fluids and synovial membranes of RA patients. The major cell types producing ORM2 were synovial macrophages and fibroblast-like synoviocytes (FLSs) from RA patients. Recombinant ORM2 robustly increased IL-6, TNF-α, CXCL8 (IL-8), and CCL2 production by RA macrophages and FLSs via the NF-κB and p38 MAPK pathways. Interestingly, glycophorin C, a membrane protein for determining erythrocyte shape, was the receptor for ORM2. Intra-articular injection of ORM2 increased the severity of arthritis in mice and accelerated the infiltration of macrophages into the affected joints. Moreover, circulating ORM2 levels correlated with RA activity and radiographic progression. In conclusion, the acute phase protein ORM2 can directly increase the production of proinflammatory mediators and promote chronic arthritis in mice, suggesting that ORM2 could be a new therapeutic target for RA.

Abstract 1188: Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast cancer

Abstract Metastatic triple-negative breast cancer (mTNBC) currently has the poorest prognosis among subtypes, with limited treatment options due to molecular characteristics that render conventional targeted therapies ineffective. Immunotherapy, specifically anti-PD-(L)1 (ICIs), has revolutionized certain cancer treatments, but its efficacy in mTNBC has been disappointing. To enhance tumor responses to ICIs, combining them with immunogenic chemotherapy has been explored. Studies like IMpassion130 and KEYNOTE 522 indicate benefits of chemo-immunotherapy based on taxanes, especially in PD-L1+ tumors. PD-L1 expression often correlates with TILs infiltration, particularly cytotoxic T lymphocytes (CTLs), indicating an activated immune response in the tumor. However, tumors with poor CTL infiltration, known as "Cold" phenotype, are often associated with a lack of induction of CXCR3-associated chemokines (CXCL9, 10, and 11), crucial for CTLs recruitment in the tumor microenvironment. The limited efficacy of chemo-immunotherapy in PD-L1-negative tumors may stem from chemotherapy's inability to induce immunogenic cancer cell death, CXCR3-associated chemokines, and CTL recruitment. Internal data from a 4T1 TNBC murine model demonstrated that the taxane paclitaxel failed to induce CXCL10, CTL recruitment, and sensitization to PD-L1 blockade. The preclinical research project aimed to identify a combination therapy, inspired by current clinical practices, capable of inducing CXCL10 expression and restoring CTL recruitment in the 4T1 "cold" mTNBC model. We initially screened standard TNBC chemotherapies and selected carboplatin for its in vitro potential to induce CXCL10 production and its clinical use in association with ICIs in the TNBC. Combining carboplatin with a library of over 400 targeted therapies, we identified histone deacetylase (HDAC) inhibitors as significantly enhancing CXCL10 secretion. In vivo, this combination demonstrated therapeutic additivity, increasing immune recruitment, particularly CD8+ T lymphocytes, with the therapeutic effect reduced by CXCR3 blockade. Mechanistic exploration in vitro revealed that the combination activated the type I interferon pathway, inducing CXCL10 secretion. In vivo confirmation validated the role of the type I interferon pathway and CXCL10 in the combination's efficacy. This data outlines a chemotherapy and HDAC inhibitor combination capable of inducing CTL recruitment via increased CXCL10 production. Future project phases will focus on understanding HDACi's mechanism of action on CXCL10 production and the combination's potential to sensitize tumors to ICIs. Overall, this project underscores the role of epigenetic modifications in amplifying carboplatin-induced CXCL10 secretion, potentially converting "cold" ICI-resistant tumors into a "hot" phenotype. Citation Format: Laura Kalfeist, Stacy Petit, Loick Galland, Cyriane Poirrier, Romain Aucagne, François Ghiringhelli, Sylvain Ladoire, Emeric Limagne. Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1188.

Abstract 5529: Mimicking CAFs, pial cells support leptomeningeal metastasis

Abstract Leptomeningeal metastasis (LM), the spread of cancer into cerebrospinal fluid (CSF)-filled coverings of the brain and spinal cord, is a dismal complication of cancer leading to rapid neurologic disability and death. While a diverse array of primary tumors may result in LM, recent work from our laboratory (Chi Y et al 2020 Science) demonstrates a conserved transcriptional signature, independent of primary tumor, suggesting that tumor-microenvironmental interactions play a dominant role in this pathology. We have proteomically interrogated CSF collected from breast (n = 45), lung (n = 30), and melanoma (n = 27) patients with and without LM. We detect robust expression of inflammatory cytokines in all LM+ samples, 15 of these cytokines including IL8, IL6, and CXCL1 are conserved across tumor types in the presence of LM. Because cancer cells in the CSF do not express mRNA associated with these cytokines, we hypothesized that these inflammatory cytokines may originate from the microenvironment. Within the leptomeninges, LM cells adhere to and interact with the innermost layer of the meninges, the pia mater, which contains fibroblast-like cells. Co-culture of cancer cells with pial cells induced changes in the pial cell transcriptome and secretome, leading to increased IL8, IL6, and CXCL1 production. This communication between cancer cells and pial cells is preserved in the absence of direct cell-cell contact. These pial cells support cancer cell growth in vivo, as measured by co-implantation experiments. In addition, bulk RNA sequencing of mouse pial cells shows an inflammatory signature upregulated in the presence of LM. Taken together, these observations support a cancer-associated-fibroblast like role for pial cells in the setting of LM. Our work extends the CAF phenotype beyond classical fibroblasts to include pial cells, suggesting novel therapeutic opportunities within CNS malignancies. Citation Format: Jenna Snyder, Jan Remsik, Adrienne Boire. Mimicking CAFs, pial cells support leptomeningeal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5529.

Abstract 3864: Role of NK/TAM cross-talk on T-CD8+ recruitment and efficacy of chemoimmunotherapy and MEKi combination in "cold" tumors

Abstract In patients with non-small cell lung cancer (NSCLC), double platinum-based chemotherapy (cisplatin or carboplatin and pemetrexed) combined with the anti-PD-1 antibody has shown significant clinical benefit and has become the standard of care. In the context of so-called "cold" tumors, the immunogenic capacities of chemotherapies increase danger signals (type I IFNs, CXCL9/10) and favor both infiltration of NK/T-CD8+ cells and response to anti-PD-1. Despite the therapeutic progress represented by this new combination, some patients have limited benefit from chemo-immunotherapy, suggesting that in these patients, the immunogenic capacities of chemotherapy are limited. Recently, we were able to show that KRAS oncogene signaling restricts the ability of the cisplatin/pemetrexed doublet to induce the chemokine CXCL10, lymphocyte recruitment and sensitize to PD-L1 blockade. Thus, the addition of a MEK inhibitor (MEKi) to the chemo-immunotherapy combination restores an immunogenic signal through CXCL10, enabling T-CD8+ to infiltrate the tumor more extensively, making it more sensitive to immunotherapy. Here, we unravel the essential role of NK cells in the immunological and antitumoral effect of chemo-immunotherapy plus MEKi combination in the LLC1 preclinical lung cancer model. We showed that chemo/MEKi combination favors NK cell recruitment and activation in tumor bed through both CXCL10 and NKG2D-ligand (Rae1, Mult1). Then, IFNγ-producing NK cells trigger tumor associated macrophage switch from M2 toward M1 phenotype and amplify T-CD8+ cells recruitment through CXCL9 production by macrophages. Thus, NK cell depletion, CXCR3, CXCL9 or NKG2D-neutralization restrained chemo/MEKi therapeutic efficacy. However, IFNγ also triggers NKG2A-ligand expression (Qa-1b, HLA-E in human) which restrained NK cell activation and CD8+ T cell recruitment. Thus reducing the NKG2A pathway, by using Qa-1b-/- LLC1 cells, enhances chemo-immunotherapy/MEKi anti-tumor efficacy. Similar results were obtained in preclinical “cold” CT26 colon cancer model treated by chemo-immunotherapy. Our results underline a new role for NK cell activation to trigger TAM2/TAM1 switch, CXCL9 expression and amplify CD8 T cell recruitment in “cold” tumor. In a context of resistance to chemo-immunotherapy, MEKi or ICI targeting NK cells could be an interesting way to amplify the anti-tumor immune response by modulating both TAMs biology and the recruitment of CD8+ T cells. Citation Format: Lisa Nuttin, Charlène Latour, Solène Revy, Romain Aucagne, Guilhem Lalle, François Ghiringhelli, Emeric Limagne. Role of NK/TAM cross-talk on T-CD8+ recruitment and efficacy of chemoimmunotherapy and MEKi combination in "cold" tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3864.

Abstract 5279: Sequential treatment with PARPi and WEE1i minimizes T cell DNA damage and enhances its immune response

Abstract Background: DNA damage response (DDR) targeted therapy, like PARP inhibitors (PARPi) and WEE1 inhibitors (WEE1i), have demonstrated potent anti-cancer activity and are pronounced to activate the cytosolic immunity pathway. Our previous work showed that sequential inhibition of PARP and WEE1 could kill tumor cells as effective as their concurrent application, with ameliorated toxicity to normal cells, suggesting high clinical translational potential of this regimen. However, the effect of sequential treatment on T lymphocytes and anti-cancer immunity remains largely unknown. Methods and results: T cells were treated with 25 μM olaparib, or 250 nM AZD1775, or olaparib combined with AZD1775 (concurrent group) for 48h, or olaparib for 24h followed by AZD1775 for 24h (sequential group) in vitro. AZD1775 and concurrent treatment reduced the T cell viability to 19% and 14.3%, while sequential treatment maintained the viability to 58.6%, closed to olaparib monotherapy (61.6%). And the most potent proliferation capacity accompanied with the lowest apoptosis level were both recorded after sequential treatment, manifested by the increasing of Ki67 and EdU and the decreasing of cleaved caspase-3. Reverse-phase protein arrays (RPPA) analyses identified relieved DNA damage in T cells receiving sequential treatment. Alkaline comet assay and detecting of γH2AX further confirmed that the T cell DNA damage caused by PARPi and WEE1i was obviously alleviated after sequential therapy. Furthermore, sequential treatment activated cGAS-STING pathway effectively and upregulated the production of CCL5, CXCL10 and IFNβ of downstream type I interferon response compared with monotherapy and concurrent treatment in ovarian cancer cell lines (SKOV3, OVCAR8, A2780). We also evaluated an ID8 intraperitoneal syngeneic immunocompetent mouse model. T cells isolated from tumors, ascites, spleen and peripheral blood in sequential group were presented with the highest proliferation potential and the lowest level of DNA damage. And the tumor burden of mice in sequential group was obviously subsided, which was comparable to that in concurrent group. Conclusions: This study demonstrated that targeting PARP and/or WEE1 impaired the T cell viability and led to DNA damage. However, administrating PARPi and WEE1i sequentially diminished this inhibitory effect on T cells on the premise of ensuring their anti-tumor effect. These data provide strong rational for the further investigation on exploring the clinical significance of sequential treatment with PARPi and WEE1i. Citation Format: Xiaofei Jiao, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. Sequential treatment with PARPi and WEE1i minimizes T cell DNA damage and enhances its immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5279.

Abstract 1394: The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma

Abstract The CXCL10-CXCR3 axis is known to promote tumor growth and metastasis via autocrine signaling, while it can also elicit anti-tumor responses by paracrine signaling. However, its roles are still elusive in osteosarcoma (OS), the most common malignant bone tumor in children. In this study, we utilized in vitro assays, mouse models, and gene expression analysis to characterize the roles of the CXCL10-CXCR3 axis in OS. To understand the autocrine signaling, we performed in vitro phenotypic assays on three OS cell lines with and without CXCL10. The results showed that the chemokine increased AKT phosphorylation and tumor cell migration. Using a CRISPR-based CXCR3 deletion mutant, we demonstrated that the lack of the receptor inhibited OS tumor growth and pulmonary metastasis in an orthotopic xenograft mouse model. The results indicate that the CXCL10-CXCR3 axis is sufficient and necessary to promote aggressive phenotypes in OS via autocrine signaling. Next, using gene expression datasets from two cohorts of OS patients, we showed that high expression of CXCL10 or its cognate receptor CXCR3 was associated with a better prognosis. Since CXCL10 is known to recruit CXCR3+ immune cells to fight against cancer, we further found that the expression of the T cell marker CD3D was associated with a better prognosis. These results suggest that the chemokine may also play a protective role in osteosarcoma by recruiting anti-tumor immune cells via paracrine signaling. Lastly, we have previously reported that a high circulating level of CXCL10 correlates with a poor prognosis in osteosarcoma patients. In this study, we demonstrated that increasing the circulating CXCL10 level in immunodeficient orthotopic xenograft mouse models of OS did not significantly promote the development of pulmonary metastases, suggesting circulating CXCL10 may exert its chemotactic effect mainly on immune cells, not on tumor cells. Taken together, we propose a model that tumor expression of CXCL10 promotes osteosarcoma growth and metastasis development via autocrine signaling. The increase of the local level of CXCL10 attracts CXCR3+ immune cells to the tumor site and promotes anti-tumor function via paracrine signaling. Metastatic cells colonize the lungs and cause tissue damage and inflammation, which increases the production of CXCL10 in the circulation. The higher circulating CXCL10 recruits T cells away from the primary tumor site and indicates the occurrence of metastasis and, hence, correlates with a worse outcome in patients. Our research highlights the importance of understanding the opposite effects of CXCL10 on tumor and immune cells as well as in the tumor microenvironment and circulation. Identification of the mechanistic differences in the CXCL10-CXCR3 signaling between tumor and immune cells will provide a novel therapeutic approach against tumor cells while promoting anti-tumor immune response in OS. Citation Format: Benjamin B. Gyau, Xiang Chen, Junyan Wang, Margaret A. Clement, Angela M. Major, Jun Xu, M. John Hicks, Tsz-Kwong Man. The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1394.

Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

Background. The activation of complement is involved in monocyte recruitment in tuberculous pleural effusion (TPE), while the role of the cleavage product of complement C3 in this process needs further research. Methods. The expression of complement components in pleural biopsy specimens of TPE patients was measured. The concentration of cleavage products of complement was tested in TPE by ELISA. Moreover, the colocalizations of C3b and CR1, C3d and CR3, and CXCL12 and CXCR4 in monocytes and pleural mesothelial cells (PMCs) isolated from TPE were determined by an immunofluorescent assay. Monocyte chemotaxis assay was analyzed via transwell chambers. Results. Three pathways of the complement system were activated in tuberculous pleurisy. In patients with TPE, C3 lysis was more active than peripheral blood in pleural cavity. Tuberculous protein Mpt64 and anaphylatoxin C3a could significantly promote CXCL12 production in human PMCs isolated from TPE. C3b-CR1, C3d-CR3, and CXCL12-CXCR4 were colocalized in PMCs and monocytes from TPE. The recruitment of monocytes into TPE mediated by PMCs could be inhibited by anti-CR1, anti-CR3, and anti-CXCL12 monoclonal antibodies (mAbs). Conclusions. Complement activates strongly in TPE, and PMCs induced monocytes to the pleural cavity through C3a, C3b, and C3d.

Testosterone Inhibits Secretion of the Pro-Inflammatory Chemokine CXCL1 from Astrocytes.

Astrocytes play an important role in the regulation of the inflammatory response in the CNS, e.g., in demyelinating diseases. Since the chemokine CXCL1 is known to be secreted by astrocytes and to have a pro-inflammatory effect on immune cells in the CNS, we verified the effect of testosterone on its secretion in vitro (in the astrocytic cell line DI TNC1). Testosterone reduced the increase in CXCL1 production caused by the pro-inflammatory agent lysophosphatidylcholine and restored the basal production level of CXCL1. The androgen receptor (present and functional in the studied cell line) was strongly suggested to mediate this effect-its non-steroid ligand flutamide exerted an agonist-like effect, mimicking the activity of testosterone itself on CXCL1 secretion. This novel mechanism has important implications for the known immunomodulatory effect of testosterone and potentially other androgenic hormones. It provides a potential explanation on the molecular level and shows that astrocytes are important players in inflammatory homeostasis in the CNS and its hormonal regulation. Therefore, it suggests new directions for the development of the therapeutic intervention.

Mechanical overload-induced release of extracellular mitochondrial particles from tendon cells leads to inflammation in tendinopathy

Tendinopathy is one of the most common musculoskeletal diseases, and mechanical overload is considered its primary cause. However, the underlying mechanism through which mechanical overload induces tendinopathy has not been determined. In this study, we identified for the first time that tendon cells can release extracellular mitochondria (ExtraMito) particles, a subtype of medium extracellular particles (mEPs), into the environment through a process regulated by mechanical loading. RNA sequencing systematically revealed that oxygen-related reactions, extracellular particles, and inflammation were present in diseased human tendons, suggesting that these factors play a role in the pathogenesis of tendinopathy. We simulated the disease condition by imposing a 9% strain overload on three-dimensional mouse tendon constructs in our cyclic uniaxial stretching bioreactor. The three-dimensional mouse tendon constructs under normal loading with 6% strain exhibited an extended mitochondrial network, as observed through live-cell confocal laser scanning microscopy. In contrast, mechanical overload led to a fragmented mitochondrial network. Our microscopic and immunoblot results demonstrated that mechanical loading induced tendon cells to release ExtraMito particles. Furthermore, we showed that mEPs released from tendon cells overloaded with a 9% strain (mEP9%) induced macrophage chemotaxis and increased the production of proinflammatory cytokines, including IL-6, CXCL1, and IL-18, from macrophages compared to mEP0%, mEP3%, and mEP6%. Partial depletion of the ExtraMito particles from mEP9% by magnetic-activated cell sorting significantly reduced macrophage chemotaxis. N-acetyl-L-cysteine treatment preserved the mitochondrial network in overloaded tendon cells, diminishing overload-induced macrophage chemotaxis toward mEP9%. These findings revealed a novel mechanism of tendinopathy; in an overloaded environment, ExtraMito particles convey mechanical response signals from tendon cells to the immune microenvironment, culminating in tendinopathy.

Implications of NLRP3 Suppression Using Glibenclamide and miR-223 against Colorectal Cancer.

The NLR family pyrin domain containing 3 (NLRP3) promotes the growth of colorectal cancer (CRC). However, the therapeutic effect of NLRP3 inhibition on CRC cell progression is controversial. This study comparatively investigated the therapeutic effect of a pharmacological NLRP3 inhibitor, glibenclamide (gli), and the post-translational suppression of NLRP3 by miR-223 on CRC cell progression in HCT-116 and HCT-15 cells. LPS and ATP were used to activate Gli-treated and LSB-hsa-miR-223-3p (WTmiR-223)-expressing HCT-116 cells. NLRP3.AB.pCCL.sin.cPPT.U6.miR-223-Decoy.hPGK.GFP.WPRE plasmid (DmiR-223) was the negative control for miR-223 expression. NLRP3, gasdermin D, and BAX expressions were analyzed using western blotting. Real-time PCR detected the RNA expression of autophagy-related genes ATG5, BECN1, and miR-223 in non-transfected cells. ELISA analyzed IL-1β and IL-18 in the medium. MTS-1, annexin V, wound-healing, and sphere-invasion assays were used to assess cell viability and progression. A multiplex cytokine assay detected proinflammatory cytokine secretion. LPS-ATP-activated NLRP3 produced gasdermin D cleavage, released IL-1b and IL-18, and activated cell migration and sphere invasion. In contrast, reduced cell growth, miR-223 expression, IFN-γ, CXCL10, and LIF secretion were found in cells after inflammasome activation. Both gli and WTmiR-223 induced autophagy genes ATG5 and BECN1 and reduced the NLRP3 activation and its downstream proteins. However, while gli had a limited effect on the production of IFN-γ, CXCL10, and LIF, WTmiR-223 increased the release of those cytokines. In addition, gli did not suppress cell growth, while WTmiR-223 promoted apoptosis. Notably, neither gli nor WTmiR-223 effectively prevented sphere invasion. These data suggest that, while WTmiR-223 could have a better anticancer effect in CRC compared to gli, the sole usage of miR-223-mediated NLRP3 suppression may not be sufficient to prevent CRC metastasis.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1β, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTβR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTβR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTβR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

Interferon-γ-inducible protein of 10kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cellmigration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A.Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Neuroinflammatory disease signatures in SPG11-related hereditary spastic paraplegia patients

Biallelic loss of SPG11 function constitutes the most frequent cause of complicated autosomal recessive hereditary spastic paraplegia (HSP) with thin corpus callosum, resulting in progressive multisystem neurodegeneration. While the impact of neuroinflammation is an emerging and potentially treatable aspect in neurodegenerative diseases and leukodystrophies, the role of immune cells in SPG11–HSP patients is unknown. Here, we performed a comprehensive immunological characterization of SPG11–HSP, including examination of three human postmortem brain donations, immunophenotyping of patients’ peripheral blood cells and patient-specific induced pluripotent stem cell-derived microglia-like cells (iMGL). We delineate a previously unknown role of innate immunity in SPG11–HSP. Neuropathological analysis of SPG11–HSP patient brain tissue revealed profound microgliosis in areas of neurodegeneration, downregulation of homeostatic microglial markers and cell-intrinsic accumulation of lipids and lipofuscin in IBA1+ cells. In a larger cohort of SPG11–HSP patients, the ratio of peripheral classical and intermediate monocytes was increased, along with increased serum levels of IL-6 that correlated with disease severity. Stimulation of patient-specific iMGLs with IFNγ led to increased phagocytic activity compared to control iMGL as well as increased upregulation and release of proinflammatory cytokines and chemokines, such as CXCL10. On a molecular basis, we identified increased STAT1 phosphorylation as mechanism connecting IFNγ-mediated immune hyperactivation and SPG11 loss of function. STAT1 expression was increased both in human postmortem brain tissue and in an Spg11–/– mouse model. Application of an STAT1 inhibitor decreased CXCL10 production in SPG11 iMGL and rescued their toxic effect on SPG11 neurons. Our data establish neuroinflammation as a novel disease mechanism in SPG11–HSP patients and constitute the first description of myeloid cell/ microglia activation in human SPG11–HSP. IFNγ/ STAT1-mediated neurotoxic effects of hyperreactive microglia upon SPG11 loss of function indicate that immunomodulation strategies may slow down disease progression.

P050 Paneth cell retinoid X receptor alpha drives metabolic gut inflammation

Abstract Background The rising incidence of inflammatory bowel diseases throughout the western world could be partially explained by changes in dietary behaviour such as an increased consumption of polyunsaturated fatty acids (PUFA). In previous projects we demonstrated that PUFA induce a Crohn’s disease (CD) like gut inflammation in mice lacking antioxidant GPX4 in their intestinal epithelial cells. Cellular stress within Paneth cells has been shown to drive enteritis in mice. In this project we investigated the role of the PUFA receptor and transcription factor RXRα as driver of PUFA induced metabolic gut inflammation. Methods We generated mice lacking GPX4 (GPX4ΔPC) and GPX4 as well as RXRα (GPX4/RXRαΔPC) specifically in Paneth cells and fed them and littermate controls a Western-style diet enriched with PUFA (PUFA-WD). Mice were treated with 9-cis retinoic acid, HX531 or a neutralizing CXCL1 antibody. MODE-K cells (murine IECs) were stimulated with PUFA and were analysed by biochemical methods including ELISA, qPCR, ChIP and RNA sequencing for mechanistical workup in vitro. Results When stimulated with PUFA, GPX4 deficient IECs showed an increased activation of RXRα as well as an increased transcription and production of neutrophil attracting chemokine CXCL1, the murine homologue of IL-8. This could be attenuated by ablation of RXRα or treatment with HX531, an RXRα antagonist. By using ChIP we can demonstrate that RXRα is directly binding to the promoter region of CXCL1 upon PUFA stimulation, thereby regulating its transcription. GPX4ΔPC mice develop a CD like enteritis with mucosal to submucosal infiltration of neutrophils, macrophages, B and T cells. GPX4/RXRαΔPC mice were protected from PUFA induced enteritis and gut inflammation could be significantly ameliorated by treatment of GPX4ΔPC mice with RXRα antagonist HX531 or a neutralizing CXCL1 antibody after a PUFA-WD challenge. Moreover 9-cis retinoic acid treatment dose dependently blunted CXCL1 response and attenuated PUFA induced intestinal inflammation in GPX4ΔPC mice, thus indicating a competitive binding of PUFA to RXRα. Conclusion Our findings reveal, that PUFA binding to RXRα within Paneth cells regulates the transcription of cytokines and is crucially involved in the development of diet induced metabolic gut inflammation. Future studies are now warranted to prove if RXRα could be a promising new druggable target in human CD patients.

Impaired activation of plasmacytoid dendritic cells via toll-like receptor 7/9 and STING is mediated by melanoma-derived immunosuppressive cytokines and metabolic drift.

Plasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production. The activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer. Based on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape. These findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

TRIM26 alleviates fatal immunopathology by regulating inflammatory neutrophil infiltration during Candida infection.

Fungal infections have emerged as a major concern among immunocompromised patients, causing approximately 2 million deaths each year worldwide. However, the regulatory mechanisms underlying antifungal immunity remain elusive and require further investigation. The E3 ligase Trim26 belongs to the tripartite motif (Trim) protein family, which is involved in various biological processes, including cell proliferation, antiviral innate immunity, and inflammatory responses. Herein, we report that Trim26 exerts protective antifungal immune functions after fungal infection. Trim26-deficient mice are more susceptible to fungemia than their wild-type counterparts. Mechanistically, Trim26 restricts inflammatory neutrophils infiltration and limits proinflammatory cytokine production, which can attenuate kidney fungal load and renal damage during Candida infection. Trim26-deficient neutrophils showed higher proinflammatory cytokine expression and impaired fungicidal activity. We further demonstrated that excessive neutrophils infiltration in the kidney was because of the increased production of chemokines CXCL1 and CXCL2, which are mainly synthesized in the macrophages or dendritic cells of Trim26-deficient mice after Candida albicans infections. Together, our study findings unraveled the vital role of Trim26 in regulating antifungal immunity through the regulation of inflammatory neutrophils infiltration and proinflammatory cytokine and chemokine expression during candidiasis.

Role of Small Airway Epithelial-Mesenchymal Transition and CXCL13 in Pulmonary Lymphoid Follicle Formation in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder characterized by inflammation and airway remodeling. Lymphoid follicles play a crucial role in acquired immunity and the development of COPD. However, the precise mechanisms of lymphoid follicle formation in COPD and the effects of cigarette smoke (CS) exposure on this process remain unclear. Epithelial-mesenchymal transition (EMT) is implicated in the progression of COPD and may serves as a source of stromal cells that produce chemokines crucial for lymphoid follicle formation. This study aims to clarify the contributions and mechanisms of EMT in lymphoid follicle genesis in COPD, focusing specifically on the role of CXCL13. Lung tissue samples were obtained from patients with COPD, smokers, and non-smokers. Immunohistochemistry was performed to assess the lymphoid follicles, EMT-related markers, and CXCL13 expression. In vitro experiments were conducted using CS extract (CSE)-stimulated immortalized human bronchial epithelial cells (iHBECs) to induce EMT. The expression of EMT-related markers and CXCL13 in CSE-stimulated iHBECs was analyzed using Western blotting, real-time PCR, and immunofluorescence staining. The effect of an EMT inhibitor on CXCL13 expression was also examined. Patients with COPD and lymphoid follicles exhibited significantly lower forced expiratory volume in 1 s (% predicted) values than those without lymphoid follicles. Enhanced EMT changes were observed in patients with COPD and lymphoid follicles. Increased EMT-related markers and CXCL13 expression were observed in CSE-stimulated iHBECs, and CXCL13 expression gradually increased over time. Inhibiting EMT downregulated CXCL13 expression in iHBECs. Lymphoid follicles are associated with enhanced EMT in COPD. EMT may act as a key driver of the adaptive immune response in COPD by promoting a microenvironment conducive to lymphoid follicles formation through the production of CXCL13. This study provides valuable insights into the mechanisms underlying lymphoid follicle formation in COPD and identifies potential therapeutic targets.