Chemokine CCL2 Research Articles

Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9–11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman’s correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

Novel chemokine biomarkers in melanoma†.

The melanoma tumor microenvironment is a complex milieu of cancer, inflammatory, and stromal cells. In this context, chemokines play a pivotal role in recruiting inflammatory cells and influence the tumor, exerting both pro-tumorigenic and anti-tumoral roles. Interactions between these cells is what ultimately hold together and transform the tumor into an efficient machine. A recent study found that chemokines CCL8, CCL15, and CCL20 were upregulated in melanoma cells when co-cultured with macrophages and were associated with poor survival rates. CCL8 and CCL15 also stimulated melanoma cell growth, invasion, and metastasis, and were highly expressed in tumors prone to metastasize, suggesting these chemokines are attractive and independent biomarkers. Understanding the intricated interactions within the tumor microenvironment could lead to prognostic biomarkers and to the development of new therapeutic strategies for melanoma. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mononuclear phagocyte sub-types in vitro display diverse transcriptional responses to dust mite exposure

Mononuclear phagocytes (MNP), including macrophages and dendritic cells form an essential component of primary responses to environmental hazards and toxic exposures. This is particularly important in disease conditions such as asthma and allergic airway disease, where many different cell types are present. In this study, we differentiated CD34+ haematopoietic stem cells towards different populations of MNP in an effort to understand how different cell subtypes present in inflammatory disease microenvironments respond to the common allergen house dust mite (HDM). Using single cell mRNA sequencing, we demonstrate that macrophage subtypes MCSPP1+ and MLCMARCO+ display different patterns of gene expression after HDM challenge, noted especially for the chemokines CXCL5, CXCL8, CCL5 and CCL15. MLCCD206Hi alternatively activated macrophages displayed the greatest changes in expression, while neutrophil and monocyte populations did not respond. Further work investigated how pollutant diesel exhaust particles could modify these transcriptional responses and revealed that CXC but not CC type chemokines were further upregulated. Through the use of diesel particles with adsorbed material removed, we suggest that soluble pollutants on these particles are the active constituents responsible for the modifying effects on HDM. This study highlights that environmental exposures may influence tissue responses dependent on which MNP cell type is present, and that these should be considerations when modelling such events in vitro. Understanding the nuanced responsiveness of different immune cell types to allergen and pollutant exposure also contributes to a better understanding of how these exposures influence the development and exacerbation of human disease.

Topical application of a CCL22-binding aptamer suppresses contact allergy

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nucleotide DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.

Grooved Microneedle Patch Augments Adoptive T Cell Therapy Against Solid Tumors via Diverting Regulatory T Cells.

The efficacy of adoptive T cell therapy (ACT) for the treatment of solid tumors remains challenging. In addition to the poor infiltration of effector T (Teff) cells limited by the physical barrier surrounding the solid tumor, another major obstacle is the extensive infiltration of regulatory T (Treg) cells, a major immunosuppressive immune cell subset, in the tumor microenvironment. Here, this work develops a grooved microneedle patch for augmenting ACT, aiming to simultaneously overcome physical and immunosuppressive barriers. The microneedles are engineered through an ice-templated method to generate the grooved structure for sufficient T-cell loading. In addition, with the surface modification of chemokine CCL22, the MNs could not only directly deliver tumor-specific T cells into solid tumors through physical penetration, but also specifically divert Treg cells from the tumor microenvironment to the surface of the microneedles via a cytokine concentration gradient, leading to an increase in the ratio of Teff cells/Treg cells in a mouse melanoma model. Consequently, this local delivery strategy of both T cell receptor T cells and chimeric antigen receptor T cells via the CCL22-modified grooved microneedles as a local niche could significantly enhance the antitumor efficacy and reduce the on-target off-tumor toxicity of ACT.

Circulating Chemokines and Short- and Long-Term Outcomes After Ischemic Stroke

AbstractChemokines are vital in post-cerebral ischemia inflammatory reactions. We investigate the possible relationship between plasma chemokines and short-term and long-term outcomes after stroke. This study included 235 patients (median age, 72 years; 49.8% female) suffering from ischemic stroke, or transient ischemic attack admitted to the hospital within 24 h of onset. We evaluated chemokines CCL2, CCL5, CXCL8, CXCL9, and CXCL10 in plasma samples collected upon admission. Further, we assessed functional outcomes at 3- and 12-months, all-cause fatality over 5 years, and episodes of delirium within the first 7 days of admission. Multivariate analysis revealed an association between higher CXCL10 levels and an increased risk of poor functional outcomes at 3 months (OR: 3.02, 95%CI: 1.22–7.46, p = 0.016) and 12 months (OR: 2.32, 95%CI: 1.03–5.26, p = 0.043), as well as an increased death risk (HR: 1.79, 95%CI: 1.04–3.07, p = 0.036). High CXCL8 levels independently predicted poor functional outcomes at 12 months (OR: 2.69, 95%CI: 1.39–6.31, p = 0.005) and a higher 5-year case fatality rate (HR: 1.90, 95%CI: 1.23–2.93, p = 0.004). Elevated CXCL9 levels also predicted unfavourable functional outcomes at 12 months (OR: 2.45, 95%CI: 1.07–5.61, p = 0.034). In univariate analysis, increased levels of CXCL8, CXCL9, and CXCL10 showed an association with delirium, although this link was not evident in the multivariate analysis. Plasma CXCL8 and CXCL10 show potential as prognostic biomarkers for stroke outcomes and as therapeutic targets suitable for reverse translation.

Race-Related Differences in Sipuleucel-T Response among Men with Metastatic Castrate-Resistant Prostate Cancer.

Our novel findings of higher expression of co-stimulatory receptor ICOS on CD4+ and CD8+ T cells in African American patients with metastatic castrate-resistant prostate cancer (mCRPC) prior and post-sipuleucel-T suggest activation of CD4+ and CD8+ T cells. The data indicate that racial differences observed in these and other immune correlates before and after sipuleucel-T warrant additional investigation to further our understanding of the immune system in African American men and other men with mCRPC.

CCL22 Induces the Polarization of Immature Dendritic Cells into Tolerogenic Dendritic Cells in Radiation-Induced Lung Injury through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 Signaling Pathway.

Recruitment of immune cells to the injury site plays a pivotal role in the pathology of radiation-associated diseases. In this study, we investigated the impact of the chemokine CCL22 released from alveolar type II epithelial (AT2) cells after irradiation on the recruitment and functional changes of dendritic cells (DCs) in the development of radiation-induced lung injury (RILI). By examining changes in CCL22 protein levels in lung tissue of C57BL/6N mice with RILI, we discovered that ionizing radiation increased CCL22 expression in irradiated alveolar AT2 cells, as did MLE-12 cells after irradiation. A transwell migration assay revealed that CCL22 promoted the migration of CCR4-positive DCs to the injury site, which explained the migration of pulmonary CCR4-positive DCs in RILI mice invivo. Coculture experiments demonstrated that, consistent with the response of regulatory T cells in the lung tissue of RILI mice, exogenous CCL22-induced DCs promoted regulatory T cell proliferation. Mechanistically, we demonstrated that Dectin2 and Nr4a2 are key targets in the CCL22 signaling pathway, which was confirmed in pulmonary DCs of RILI mice. As a result, CCL22 upregulated the expression of PD-L1, IL-6, and IL-10 in DCs. Consequently, we identified a mechanism in which CCL22 induced DC tolerance through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 pathway. Collectively, these findings demonstrated that ionizing radiation stimulates the expression of CCL22 in AT2 cells to recruit DCs to the injury site and further polarizes them into a tolerant subgroup of CCL22 DCs to regulate lung immunity, ultimately providing potential therapeutic targets for DC-mediated RILI.

Mucoadhesive chitosan-catechol as an efficient vaccine delivery system for intranasal immunization

The mucosal barrier and scavenging effect of the mucosal layer are two main obstacles in inducing mucosal immunization. To overcome these obstacles, we synthesized a bio-inspired mucoadhesive material, chitosan-catechol (ChiC), for surface modification of inactive porcine epidemic diarrhea virus (PEDV). Studies have revealed that PEDV particles can be facilely and mildly modified by Chi-C forming Chi-C-PEDV nanoparticles (Chic-Ps) through the covalent and electrostatic bond, which effectively prolongs the retention time of PEDV in the nasal mucosa. The cell co-culture model demonstrated that Chic-Ps exhibit enhanced recruitment of dendritic cells via the secretion of stimulating chemokine CCL20 and improving antigen permeability by disruption the distribution of ZO-1 protein in epithelial cells. Additionally, the flow cytometry (FCM) analysis revealed that Chic-Ps facilitate trafficking to lymph nodes and induce stronger cellular and humoral immune responses compared to unmodified PEDV. Notably, Chic-Ps induced a higher level of PEDV neutralizing antibody was induced by Chic-Ps in the nasal washes, as confirmed by a plaque reduction neutralization test. These results demonstrate that Chi-C is a promising nasal delivery system for vaccines. Proof of principle was obtained for inactivated PEDV, but similar delivery mechanisms could be applied in other vaccines when intranasal administration is needed.

Fusobacterium nucleatum to promote esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of macrophages to the tumour micro environment.

e14668 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignant tumor in China. Recent studies have shown that Fusobacterium nucleatum ( F. nucleatum) promotes ESCC progression through various mechanisms. One of them is about changing the tumor micro- environment through cytokine–cytokine receptor interaction. Methods: From January 2022 to June 2023, a total of 34 patients with ESCC from 4th Hospital of Hebei Medical University thoracic surgery were enrolled. All of the patients did not apply any neoadjuvant therapy before and received radical resection. Real-time reverse transcriptase - PCR (RT-PCR) were performed to examine the expressions of F. nucleatum in tumor tissues and para-tumor tissue. According to the CT values of the RT-PCR results, the level of the F. nucleatum infection could be separated in two groups - positive group and negative group. Immunohistochemistry (IHC) staining were used to examine the expressions of chemokine CCL20 in both groups. Immunofluorescence (IF) was characterised homing receptors CCR6 on CD206+ macrophages. In addition, transwell assay was carried on to quantify macrophages migration ability towards chemokines CCL20 in vitro. Results: F. nucleatum DNA positivity was significantly associated with higher expression of chemokine CCL20 and accumulation of CD68+CD206+ macrophages. F. nucleatum DNA positivity was also significantly correlated with worse histopathological grading. Otherwise, there were no significant correlation between F. nucleatum infection and gender, age, tobacco or alcohol use, T or N grade, TNM stage, tumor locations, blood vessel or nerve invasion and tumor size. IHC showed that the expression of CCL20 were higher in F. nucleatum DNA positive tumor tissue. After coculture with F. nucleatum and KYSE30 cell lines in vitro, the CCL20 production by KYSE30 cells were accelerated. In addition, immunofluorescence showed that CCL20 could recruit M2-like macrophages into the tumor environment by bonding CCR6 existed on the cell surface. Furthermore, the migration ability of macrophages was distinctly elevated by CCL20 result from F. nucleatum infection. Conclusions: F. nucleatum promotes esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of M2-like macrophages to the tumor micro- environment and deteriorates the suppression of the local immune micro- environment within the tumor. Such bacteria may be a biomarker to predict the efficacy of immunotherapy in ESCC. [Table: see text]

Orally administered yeast-derived β-glucan alleviates mast cell-dependent airway hyperresponsiveness and inflammation in a murine model of asthma.

Particulate β-glucans (WGP) are natural compounds with regulatory roles in various biological processes, including tumorigenesis and inflammatory diseases such as allergic asthma. However, their impact on mast cells (MCs), contributors to airway hyperresponsiveness (AHR) and inflammation in asthma mice, remains unknown. C57BL/6 mice underwent repeated OVA sensitization without alum, followed by Ovalbumin (OVA) challenge. Mice received daily oral administration of WGP (OAW) at doses of 50 or 150 mg/kg before sensitization and challenge. We assessed airway function, lung histopathology, and pulmonary inflammatory cell composition in the airways, as well as proinflammatory cytokines and chemokines in the bronchoalveolar lavage fluid (BALF). The 150 mg/kg OAW treatment mitigated OVA-induced AHR and airway inflammation, evidenced by reduced airway reactivity to aerosolized methacholine (Mch), diminished inflammatory cell infiltration, and goblet cell hyperplasia in lung tissues. Additionally, OAW hindered the recruitment of inflammatory cells, including MCs and eosinophils, in lung tissues and BALF. OAW treatment attenuated proinflammatory tumor necrosis factor (TNF)-α and IL-6 levels in BALF. Notably, OAW significantly downregulated the expression of chemokines CCL3, CCL5, CCL20, CCL22, CXCL9, and CXCL10 in BALF. These results highlight OAW's robust anti-inflammatory properties, suggesting potential benefits in treating MC-dependent AHR and allergic inflammation by influencing inflammatory cell infiltration and regulating proinflammatory cytokines and chemokines in the airways.

Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

The choroid plexus (ChP) serves as the principal origin of cerebrospinal fluid (CSF). CSF hypersecretion due to ChP inflammation has emerged as an important pathogenesis of hydrocephalus recently. Nevertheless, the precise mechanisms of ChP inflammation and the ensuing CSF hypersecretion in hydrocephalus remain ill-defined. In the present study, we elucidate the critical role of macrophages in the pathogenesis of ChP inflammation. Specifically, we identify the chemokine CCL2, released by ChP epithelial cells, recruits CCR2+ monocytes to the ChP thereby inciting hydrocephalus pathogenesis. The accumulated ChP macrophages increase the inflammation in ChP epithelial cells through TNF-α/TNFR1/NF-κB signaling cascade, thereby leading to CSF hypersecretion. Strikingly, augmentation of ChP‒CCL2 using an adeno-associated viral approach (AAV) exacerbates macrophage recruitment, activation, and ventriculomegaly in rat PHH models. Systemic application of Bindarit, a specific CCL2 inhibitor, significantly inhibits ChP macrophage infiltration and activation and reduces CSF secretion rate. Furthermore, the administration of CCR2 antagonist (INCB 3284) reduces ChP macrophage accumulation and ventriculomegaly. This study not only unveils the ChP CCL2‒CCR2 signaling in the pathophysiology of hydrocephalus but also unveils Bindarit as a promising therapeutic choice for the management of posthemorrhagic hydrocephalus.

Dissecting the MUC5AC/ANXA2 signaling axis: implications for brain metastasis in lung adenocarcinoma

Non-small cell lung carcinoma (NSCLC) exhibits a heightened propensity for brain metastasis, posing a significant clinical challenge. Mucin 5ac (MUC5AC) plays a pivotal role in the development of lung adenocarcinoma (LUAD); however, its role in causing brain metastases remains unknown. In this study, we aimed to investigate the contribution of MUC5AC to brain metastasis in patients with LUAD utilizing various brain metastasis models. Our findings revealed a substantial increase in the MUC5AC level in LUAD brain metastases (LUAD-BrM) samples and brain-tropic cell lines compared to primary samples or parental control cell lines. Intriguingly, depletion of MUC5AC in brain-tropic cells led to significant reductions in intracranial metastasis and tumor growth, and improved survival following intracardiac injection, in contrast to the observations in the control groups. Proteomic analysis revealed that mechanistically, MUC5AC depletion resulted in decreased expression of metastasis-associated molecules. There were increases in epithelial-to-mesenchymal transition, tumor invasiveness, and metastasis phenotypes in tumors with high MUC5AC expression. Furthermore, immunoprecipitation and proteomic analysis revealed a novel interaction of MUC5AC with Annexin A2 (ANXA2), which activated downstream matrix metalloproteases and facilitated extracellular matrix degradation to promote metastasis. Disrupting MUC5AC-ANXA2 signaling with a peptide inhibitor effectively abrogated the metastatic process. Additionally, treatment of tumor cells with an astrocyte-conditioned medium or the chemokine CCL2 resulted in upregulation of MUC5AC expression and enhanced brain colonization. In summary, our study demonstrates that the MUC5AC/ANXA2 signaling axis promotes brain metastasis, suggesting a potential therapeutic paradigm for LUAD patients with high MUC5AC expression.

Improved Immunotherapy Outcomes via Cuproptosis Upregulation of HLA-DRA Expression: Promoting the Aggregation of CD4+ and CD8+T Lymphocytes in Clear Cell Renal Cell Carcinoma.

Immunotherapy has shown promising clinical results in clear cell renal cell carcinoma (ccRCC), but low clinical target response rates due to dysfunction of the major histocompatibility complex (MHC) and an inhibitory tumor immune microenvironment (TIME) have largely limited the associated clinical benefits. In the present study, we explored the feasibility of enhancing tumor-specific-MHC-II-HLA-DRA expression, counteracting the TIME's suppressive effects, thereby improving the sensitivity of immune checkpoint inhibitor (ICI) therapy from the standpoint of cuproptosis. Immunohistochemical staining and in vitro experiments validated the expression of HLA-DRA in ccRCC and its positive impact on ICI therapy. Subsequently, we observed that cuproptosis upregulated HLA-DRA expression in a dose-dependent manner, further confirming the link between cuproptosis and HLA-DRA. In vivo experiments showed that cuproptosis increased the sensitivity to ICI treatment, and implementing cuproptosis alongside anti-PD-1 treatment curtailed tumor growth. Mechanistically, cuproptosis upregulates HLA-DRA expression at the transcriptional level in a dose-dependent manner by inducing the production of reactive oxygen species; high levels of HLA-DRA promote the expression of chemokines CCL5, CXCL9, and CXCL10 in the TIME, inhibiting the development of a pro-tumor microenvironment by promoting the infiltration of CD4+T and CD8+T cells, thereby synergizing ICI therapy and exerting anti-tumor effects. Taken together, this work highlights the role of cuproptosis in mediating TIME remodeling and synergistic immunotherapy, providing new evidence that cuproptosis can evoke effective anti-tumor immune responses.

#1290 Adverse proinflammatory responses in the kidney after FGF23 administration in three murine kidney disease models

Abstract Background and Aims The hormone FGF23 is a biomarker associated with morbidity and mortality in individuals with and without kidney disease, but the role of FGF23 excess as a pathogenic factor is incompletely understood. Here, we investigated renal FGF23 signaling in kidney disease models. Method This study used three independent disease models: i) Male C57BL/6 mice (n = 16) were treated with Freud's adjuvant and nephrotoxic serum (NTS) to induce anti-glomerular basement membrane (anti-GBM) disease. ii) Female BALB/c mice (n = 8) were treated with adriamycin (doxorubicin) 10.5 mg/kg to induce focal and segmental glomerulosclerosis (FSGS). iii) Male DBA/2J mice (n = 20) were fed a diet containing adenine 0.2%. Control mice of the respective same strain and sex received vehicle injections. Mice of the anti-GBM and FSGS models and controls received intravenous injections of recombinant FGF23 1 µg or vehicle for six consecutive days (anti-GBM) or once (FSGS), with dissection 24 h after the last injection. Mice of the adenine model were dissected after 15 weeks. Following dissection, ex vivo precision-cut kidney slices (PCKS) were obtained from n = 4 mice and maintained for 24 at 37°C, 95% O2 and 5% CO2, followed by a 1 h treatment with recombinant FGF23 200 ng/ml or vehicle. Glomerular filtration rate (GFR) or renal retention parameters in serum and albuminuria were assessed. Kidneys underwent histological assessment using hematoxylin & eosin or Masson's trichrome. Renal RNA was isolated for bulk RNAseq. Differential gene expression was assessed to determine the effect of FGF23 treatment in disease-free controls, in each disease model, and the interaction between FGF23 X disease effect for the anti-GBM and FSGS groups. Gene set enrichment analysis was performed with a focus on the immune system. Results Anti-GBM mice showed a 45 ± 27% GFR decline (mean ± SD) compared to baseline, whereas controls showed an GFR increase of 11 ± 36% (p < 0.01). Kidney histology revealed an interstitial infiltrate. The FSGS model showed elevated serum creatinine and albuminuria, and hypercellularity in some glomeruli. Mice of the adenine model showed progressively increased blood urea nitrogen concentrations starting after 2 weeks, up to a 3-fold increase at week 14. Histology revealed interstitial fibrosis and tubular atrophy. Bulk RNAseq revealed proinflammatory transcriptional signatures after FGF23 treatment in all disease models, regardless of which timepoint or model: In the anti-GBM model, Hallmark gene sets of inflammatory response, interferon-α response, interferon-β response and allograft rejection were significantly increased after 6 days of FGF23 treatment, whereas disease-free controls showed no significantly changed immune gene sets after 6 days of FGF23. In FSGS mice, none of the Hallmark gene sets were changed after 1 FGF23 injection, but top individual gene expression affected by 1 FGF23 injection were an increased proinflammatory transcription factor Cebpb and an increased DNA damage transcript Dclre1b. Finally, the PCKS of adenine model revealed no significant Hallmark gene sets, but the two genes with differential expression were a proinflammatory macrophage/fibroblast chemokine Ccl4 (increased) and an immunoglobulin heavy variable Ighv2-2 (decreased). Conclusion FGF23-driven patterns of proinflammatory gene expression were observed in three experimental models of kidney disease, both in vivo and ex vivo assessment of FGF23 signaling, thus excluding effects of FGF23 on other organs. As these findings were limited to transcriptional analyses, the present data warrant further confirmation and exploration of FGF23 as a pathogenic agent in kidney disease.

#1449 Maturation of tertiary lymphoid structures in progressive kidney damage due to chronic phosphate load

Abstract Background and Aims We have recently shown that a high phosphate diet (HPD) in mice leads to an increase in FGF23 levels and causes progressive tubular damage and interstitial fibrosis with increased accumulation of macrophages and concomitant perivascular immune cell aggregates in the corticomedullary zone. Such tertiary lymphoid structures (TLS) can develop as a result of chronic inflammation and have already been described in lupus nephritis and IgA nephropathy, among others. In our model, the question arose to what extent damage to the proximal tubule is temporally associated with the development and maturation of TLS and which factors favor TLS development. Method Wild-type mice were fed HPD for 1, 2, 3, 4 and 6 months and the kidneys of each were compared with those of mice on a normal phosphate diet (NPD). To evaluate the effect of high FGF23 ± phosphate, Fgf23 was overexpressed in mice on NPD and HPD using an adeno-associated virus (AAV-Fgf23), and hypophosphatemic Hyp mice were used. Results An increased tubular damage score was seen after only two months of HPD. In parallel, perivascular immune cell clusters developed in the corticomedullary zone and in the renal cortex, which were characterized by aggregates of CD45R+ B cells and CD3+ T cells, as well as LYVE1+ lymphatic vessels after three months of HPD and were accompanied by increased expression of venous markers (Plvap, Aplnr, EphB4) and cell adhesion molecules (Sell, Vcam1, Madcam1). Further analyses showed a time-dependent induction of the immunofibroblast-derived chemokines Cxcl13 and Ccl19, as well as the lymphotoxins Lta and Ltb, which are important for the differentiation of immunofibroblasts into follicular dendritic cells (FDC) and follicular reticular cells (FRC). Already after four months of HPD, proliferating B-cell clusters with FDCs, FRCs located in T-cell clusters, podoplanin+ networks, high endothelial venules, plasma cells and increased IgD synthesis indicated fully mature TLS, while tubular damage and fibrosis continued to increase until six months of HPD. Compared to AAV-Fgf23 mice on NPD, only AAV-Fgf23 animals on HPD developed tubular damage with fibrosis and fully mature TLS. Hypophosphatemic Hyp mice with high FGF23 showed neither signs of tubular damage nor the development of TLS. Conclusion The development of renal TLS due to chronic high phosphate load coincides with the development of tubular damage, with earlier maturation of TLS and progression of tubular damage.

Rabies Virus Infection Causes Pyroptosis of Neuronal Cells.

Rabies virus (RABV) is a neurotropic virus that causes fatal neurological disease, raising serious public health issues and attracting extensive attention in society. To elucidate the molecular mechanism of RABV-induced neuronal damage, we used hematoxylin-eosin staining, transmission electron microscopy, transcriptomics analysis, and immune response factor testing to investigate RABV-infected neurons. We successfully isolated the neurons from murine brains. The specificity of the isolated neurons was identified by a monoclonal antibody, and the viability of the neurons was 83.53-95.0%. We confirmed that RABV infection induced serious damage to the neurons according to histochemistry and transmission electron microscope (TEM) scanning. In addition, the transcriptomics analysis suggested that multiple genes related to the pyroptosis pathway were significantly upregulated, including gasdermin D (Gsdmd), Nlrp3, caspase-1, and IL-1β, as well as the chemokine genes Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl12, and Cxcl10. We next verified this finding in the brains of mice infected with the rRC-HL, GX074, and challenge virus standard strain-24 (CVS-24) strains of RABV. Importantly, we found that the expression level of the Gsdmd protein was significantly upregulated in the neurons infected with different RABV strains and ranged from 691.1 to 5764.96 pg/mL, while the basal level of mock-infected neurons was less than 100 pg/mL. Taken together, our findings suggest that Gsdmd-induced pyroptosis is involved in the neuron damage caused by RABV infection.