Interstitial Macrophages Research Articles

Unravelling the role of SOCS3 in aortic valve stenosis

Abstract Background Calcific aortic valve stenosis (CAVD) is the most prevalent heart valve disease worldwide and, in advanced stages, leads to clinical deterioration and poor prognosis. Until now, no medical treatment is available. Thought to be a merely degenerative disease, we now know that disease initiation and progression are actively regulated by immune cell infiltration, chronic inflammation, osteogenic differentiation of valvular interstitial cells (VIC) and endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VEC). Suppressor of Cytokine Signaling 3 (SOCS3) is known to be a key regulator of inflammation. It promotes the polarisation of macrophages to an inflammatory phenotype and thus, contributes to disease progression in atherosclerosis. In CAVD, not only inflammation but also heterotopic bone formation leads to the thickening and obstruction of the aortic valve cusps and as SOCS3 is one of the main regulators of physiological bone formation, it could play a critical role in the progression of CAVD. Purpose We hypothesize that SOCS3 plays a crucial role during calcification of the aortic valve through regulation of EndMT, calcification of valvular interstitial cells and macrophage polarization. Methods and results In initial screening experiments, we investigated SOCS3-protein expression in explanted human aortic valves from patients undergoing surgical aortic valve replacement. SOCS3 is expressed in both stenotic and non-stenotic valve tissues. Immunofluorescence-staining of human aortic valves shows the co-localisation of SOCS3 with the interstitial cell-marker Vimentin and SOCS1, which is known to regulate macrophage-polarisation together with SOCS3 (Fig. 1). Staining of the macrophage-marker CD68 revealed its co-localisation with calcified areas of the aortic valve cusp. We validated our findings in our in vitro model of VIC calcification using two different calcifying conditions. Upregulation of RUNX2 and BMP2 verified successful osteogenic differentiation of VICs. During osteogenic differentiation for 7 days, SOCS3 and SOCS1 are significantly upregulated (Fig. 2A). We were able to induce EndMT by stimulating VEC with either TNFα or TGFβ/IL1β in vitro. We show that EndMT leads to the loss of endothelial cell markers like eNOS and VWF and to an upregulation of interstitial cell markers (such as Vimentin and α-SMA) and markers of EndMT (SNAI2). During EndMT, we were also able to observe a significant upregulation of SOCS1 and SOCS3 after 7 days (Fig. 2B). Ongoing knockdown experiments will help to elucidate the role of SOCS1 and SOCS3 during initiation and progression of CAVD. Conclusion We aim towards a better understanding of SOCS3 and its role in inflammation and calcification as hallmarks of the pathogenesis of CAVD. Since Zoledronic acid has been shown to induce a decreased expression of SOCS3 in macrophages, this might present as a possible treatment strategy to target SOCS3 therapeutically. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Combining Multiple Biomarkers to Evaluate the Environmental Stress in Cyprinid Populations

The Vilariça River was restored in order to improve the fish habitat due to the construction of dams. However, due to the pressure that agriculture exerts on water resources, it is crucial to evaluate its consequences on fish health from a histological perspective. The gonad histopathological changes of two cyprinid species were assessed microscopically and evaluated with semi-quantitative analysis. Histopathological changes in the gonad, gill, and liver were used as biomarkers in the determination of the Integrated Biomarker Response (IBR), as well as to evaluate the histopathological scores between seasons. The observation of the gonad histopathological changes showed that interstitial fibrosis and macrophage aggregates were present exclusively in the Summer and more prevalent in males of large size. In addition, the histopathological scores of the three organs were higher in the Summer. In terms of the severity degree, 98% and 68% of the fish showed pronounced and severe histopathological changes in the gill and liver, respectively, while 28% of the fish showed slight histopathological changes in the gonad. The IBR showed higher values in the Summer and in the middle stream and downstream, which is in agreement with the metal concentrations. Thus, this work showed a relationship between the anomalies present in the fish organs and the quality of the water (classified as polluted). Therefore, minimization measures are presented, such as improving the irrigation methods, preserving the vegetation cover; promoting reforestation in degraded and burned areas; and improving the riparian vegetation.

Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation.

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy of the mouse lung, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.

The emerging roles of interstitial macrophages in pulmonary fibrosis: A perspective from scRNA-seq analyses.

Pulmonary fibrosis is an irreversible and progressive disease affecting the lungs, and the etiology remains poorly understood. This disease can be lethal and currently has no specific clinical therapeutic regimen. Macrophages, the most common type of immune cell in the lungs, have been reported to play a key role in the pathogenesis of fibrotic disease. The lung macrophage population is mostly composed of alveolar macrophages and interstitial macrophages, both of which have not been thoroughly studied in the pathogenesis of lung fibrosis. Interstitial macrophages have recently been recognised for their participation in lung fibrosis due to new technology arising from a combination of bioinformatics and single-cell RNA sequencing analysis. This paper reviews recent developments regarding lung macrophage classification and summarizes the origin and replenishment of interstitial macrophages and their function in pulmonary fibrosis.

Increased Density of Growth Differentiation Factor-15+ Immunoreactive M1/M2 Macrophages in Prostate Cancer of Different Gleason Scores Compared with Benign Prostate Hyperplasia.

Simple SummaryProstate cancer (PCa) is the second most diagnosed cancer and cause of death in men worldwide. The main challenge is to discover biomarkers for malignancy to guide the physician towards optimized diagnosis and therapy. There is recent evidence that growth differentiation factor-15 (GDF-15) is elevated in cancer patients. Therefore, we aimed to decipher GDF-15+ cell types and their density in biopsies of human PCa patients with Gleason score (GS)6–9 and benign prostate hyperplasia (BPH). Here we show that the density of GDF-15+ cells, mainly identified as interstitial macrophages (MΦ), was higher in GS6–9 than in BPH, and, thus, GDF-15 is intended to differentiate patients with high GS vs. BPH, as well as GS6 vs. GS7 (or even with higher malignancy). Some GDF-15+ MΦ showed a transepithelial migration into the glandular lumen and, thus, might be used for measurement in urine/semen. Taken together, GDF-15 is proposed as a novel tool to diagnose PCa vs. BPH or malignancy (GS6 vs. higher GS) and as a potential target for anti-tumor therapy. GDF-15 in seminal plasma and/or urine could be utilized as a non-invasive biomarker of PCa as compared to BPH.Although growth differentiation factor-15 (GDF-15) is highly expressed in PCa, its role in the development and progression of PCa is unclear. The present study aims to determine the density of GDF-15+ cells and immune cells (M1-/M2 macrophages [MΦ], lymphocytes) in PCa of different Gleason scores (GS) compared to BPH. Immunohistochemistry and double immunofluorescence were performed on paraffin-embedded human PCa and BPH biopsies with antibodies directed against GDF-15, CD68 (M1 MΦ), CD163 (M2 MΦ), CD4, CD8, CD19 (T /B lymphocytes), or PD-L1. PGP9.5 served as a marker for innervation and neuroendocrine cells. GDF-15+ cell density was higher in all GS than in BPH. CD68+ MΦ density in GS9 and CD163+ MΦ exceeded that in BPH. GDF-15+ cell density correlated significantly positively with CD68+ or CD163+ MΦ density in extratumoral areas. Double immunoreactive GDF-15+/CD68+ cells were found as transepithelial migrating MΦ. Stromal CD68+ MΦ lacked GDF-15+. The area of PGP9.5+ innervation was higher in GS9 than in BPH. PGP9.5+ cells, occasionally copositive for GDF-15+, also occurred in the glandular epithelium. In GS6, but not in BPH, GDF-15+, PD-L1+, and CD68+ cells were found in epithelium within luminal excrescences. The degree of extra-/intra-tumoral GDF-15 increases in M1/M2Φ is proposed to be useful to stratify progredient malignancy of PCa. GDF-15 is a potential target for anti-tumor therapy.

Comprehensive analysis of the expression of N6-methyladenosine RNA methylation regulators in pulmonary artery hypertension.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary vascular remodeling. The development of PAH involves N6-methyladenosine (m6A) modification. However, the functional role of m6A regulators in PAH and the underlying regulatory mechanisms remain unknown so far. Methods: Microarray data (GSE149713) for monocrotaline induced PAH (MCT-PAH) rat models were downloaded and screened for differentially expressed genes (DEGs) and m6A regulators. Next, we screened for differentially expressed m6A regulators in endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, interstitial macrophages, NK cells, B cells, T cells, regulatory T cells (Tregs) using scRNA sequencing data. The target DEGs of m6A regulators in ECs, SMCs, fibroblasts, and Tregs were functionally annotated using the Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In addition, the cellular interaction analysis was performed to reveal the receptor—ligand pairs regulated by m6A regulators. Pseudo-time trajectory analyses were performed and a ceRNA network of lncRNAs-miRNAs-mRNAs was constructed in SMCs. Furthermore, the RNA transcriptome sequencing data for the SMCs isolated from idiopathic PAH (IPAH) patients (GSE144274) were validated for differentially expressed m6A regulators. Moreover, the HNRNPA2B1 levels in the lung samples from PAH patients and MCT-PAH were determined using immunohistochemistry. Results: The m6A regulators were observed to be dysregulated in PAH. HNRNPA2B1expression level was increased in the PASMCs of scRNAs and IPAH patients. The target DEGs of HNRNPA2B1 were enriched in the regulation of muscle cell differentiation and vasculature development in PASMCs. The HNRNPA2B1 expression levels determined were consistent with the proliferation-related and collagen synthesis-related gene COL4A1. Moreover, the predicted transcription factors (TFs) foxd2/3 and NFκB could be involved in the regulation of HNRNPA2B1. HNRNPA2B1 might be regulating SMCs proliferation and phenotypic transition via rno-miR-330–3p/TGFβR3 and rno-miR-125a-3p/slc39a1. In addition, HNRNPA2B1 was observed to be highly expressed in the lung samples from MCT-PAH rat models and patients with PAH. Conclusion: In summary, the present study identified certain key functional m6A regulators that are involved in pulmonary vascular remodeling. The investigation of m6A patterns might be promising and provide biomarkers for diagnosis and treatment of PAH in the future.

235.5: Poly (ADP-Ribose) Polymerase (PARP) Expression in Renal Allografts Augments the Development of Epithelial-Tomesenchymal Transition (EMT), Transplant Glomerulopathy, And Interstitial Fibrosis in Pediatric Patients With Antibodymediated Rejection

Introduction: PARP activation is known to increase inflammation, but its role in developing EMT, interstitial fibrosis (IF), and transplant glomerulopathy (TG) are still unclear. Therefore we investigate the role of PARP in the development of EMT, IF, and TG in recipients with antibody-mediated rejection (AMR). Method: Expressions of tubular and glomerular PARP, α-SMA, TNF-α, TGF-β, and HLA-DR, were studied in 45 pediatric cases with AMR. Tubular α-SMA expression was noted as tubular EMT. Peritubular capillary (PTC) and interstitial leukocytes were highlighted with PARP, TNF-α, HLA-DR, and CD68. Follow-up biopsies were analyzed for IF and TG development. Results: PARP expression in tubules, glomeruli, and infiltrated leukocytes was positively correlated with PTC, glomerular, and interstitial leukocyte and macrophage infiltration (p<0.001). Tubular and glomerular PARP expression also correlated with PTC and interstitial leukocyte PARP, TNF- α, TGF-β, and HLA-DR expression (p<0.001). Tubular α-SMA expression (EMT development) positively correlated with tubular, glomerular, PTC, interstitial, PARP, TNF-α, HLA-DR, and CD68 expression (p<0.01). PARP expression increased in tubules, PTCs, interstitium, and glomeruli with the intensity of C4d expression (p<0.01). Response to rejection treatment decreases with increasing tubular, interstitial, PTC, glomerular PARP, TNF-α, HLA-DR, and CD68 expression (p<0.01). IF development time was negatively correlated with the increasing PTC and interstitial leukocyte and macrophage infiltration (p<0.001). The incidence of IF and TG development was found to increase with an increasing degree of renal PARP expression and tubular EMT development (p<0.01). Additionally, the IF development time shortened with increasing PARP, HLA-DR, TNF-α, TGF-β, α-SMA expression in inflammatory and tubular cells (P<0.01). The mean graft survival time decreased with increasing interstitial, tubular PARP, and α-SMA expression (p<0.01). The 5-year graft survival was 96% for recipients with negative tubular PARP while 60%, 19%, and 18% for recipients with grade1, grade 2, and grade 3 tubular PARP expression, respectively (p<0.001). Conclusion: Increased PARP activation leads to early graft loss by augmenting inflammation and IF by activating inflammatory signaling pathways and tubular cells myofibroblastic differentiation (EMT). Therefore, we suggest that PARP inhibitor drugs combined with immunosuppressive therapy may control inflammation and fibrosis to prevent early graft loss.

Influenza virus causes lung immunopathology through down-regulating PPARγ activity in macrophages.

Fatal influenza (flu) virus infection often activates excessive inflammatory signals, leading to multi-organ failure and death, also referred to as cytokine storm. PPARγ (Peroxisome proliferator-activated receptor gamma) agonists are well-known candidates for cytokine storm modulation. The present study identified that influenza infection reduced PPARγ expression and decreased PPARγ transcription activity in human alveolar macrophages (AMs) from different donors. Treatment with PPARγ agonist Troglitazone ameliorated virus-induced proinflammatory cytokine secretion but did not interfere with the IFN-induced antiviral pathway in human AMs. In contrast, PPARγ antagonist and knockdown of PPARγ in human AMs further enhanced virus-stimulated proinflammatory response. In a mouse model of influenza infection, flu virus dose-dependently reduced PPARγ transcriptional activity and decreased expression of PPARγ. Moreover, PPARγ agonist troglitazone significantly reduced high doses of influenza infection-induced lung pathology. In addition, flu infection reduced PPARγ expression in all mouse macrophages, including AMs, interstitial macrophages, and bone-marrow-derived macrophages but not in alveolar epithelial cells. Our results indicate that the influenza virus specifically targets the PPARγ pathway in macrophages to cause acute injury to the lung.

Senescence of alveolar epithelial cells impacts initiation and chronic phases of murine fibrosing interstitial lung disease.

Fibrosing interstitial lung disease (ILD) develops due to the impaired reparative processes following lung tissue damage. Cellular senescence has been reported to contribute to the progression of fibrosis. However, the mechanisms by which these senescent cells initiate and/or drive the progression of lung tissue fibrosis are not yet fully understood. We demonstrated that p21WAF1/CIP1- and p16INK4A-pathway-dependent senescence in type 2 alveolar epithelial cells (AEC2) were both involved in the initiation and progression of lung fibrosis in murine bleomycin (BLM)-induced ILD. p21WAF1/CIP1-senescent AEC2 emerged rapidly, as early as 1 day after the intratracheal instillation of BLM. Their number subsequently increased and persisted until the later fibrosis phase. Very few p16INK4A-senescent AEC2 emerged upon the instillation of BLM, and their increase was slower and milder than that of p21WAF1/CIP1+ AEC2. AEC2 enriched with senescent cells sorted from BLM-ILD lungs expressed senescence-associated secretory phenotype (SASP)-related genes, including Il6, Serpin1, Tnfa, Ccl2, Tgfb, and Pdgfa, at the initiation and chronic phases of fibrosis, exhibiting distinct expression patterns of magnitude that were dependent on the disease phase. Ly6C+ inflammatory monocytes increased in the lungs immediately after the instillation of BLM and interstitial macrophages increased from day 3. The expression of Acta2 and Col1a1 was upregulated as early as day 1, indicating the activation of fibroblasts. We speculated that IL-6, plasminogen activator inhibitor-1 (PAI-1), and TGF-β contributed to the accumulation of senescent cells during the progression of fibrosis in an autocrine and paracrine manner. In addition, CCL2, produced in large amounts by senescent AEC2, may have induced the infiltration of Ly6C+ inflammatory monocytes in the early phase, and TGF-β and PDGFa from senescent AEC2 may contribute to the activation of fibroblasts in the very early phases. Our study indicated that senescent AEC2 plays a role in the pathogenesis of fibrosing ILD throughout the course of the disease and provides insights into its pathogenesis, which may lead to the development of new therapeutic methods targeting senescent cells or SASP molecules.

Intratracheal Administration of Acyl Coenzyme A Acyltransferase-1 Inhibitor K-604 Reduces Pulmonary Inflammation Following Bleomycin-Induced Lung Injury.

Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage activation and failure to resolve play a role in ALI; thus, macrophage phenotype modulation is a rational target for therapeutic intervention. Large, lipid-laden macrophages have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The endoplasmic reticulum-associated enzyme acyl coenzyme A acyltransferase-1 (Acat-1/Soat1) is highly expressed in macrophages, where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat-1 will reduce macrophage activation and improve outcomes of lung injury in ITB. K-604, a selective inhibitor of Acat-1, was used to reduce cholesterol esterification and hence lipid accumulation in response to ITB. Male and female C57BL6/J mice (n = 16-21/group) were administered control, control + K-604, ITB, or ITB + K-604 on d0, control or K-604 on d3, and were sacrificed on day 7. ITB caused significant body weight loss and an increase in cholesterol accumulation in bronchoalveolar lavage cells. These changes were mitigated by Acat-1 inhibition. K-604 also significantly reduced ITB-induced alveolar thickening. Surfactant composition was normalized as indicated by a significant decrease in phospholipid: SP-B ratio in ITB+K-604 compared with ITB. K-604 administration preserved mature alveolar macrophages, decreased activation in response to ITB, and decreased the percentage mature and pro-fibrotic interstitial macrophages. These results show that inhibition of Acat-1 in the lung is associated with reduced inflammatory response to ITB-mediated lung injury.SIGNIFICANCE STATEMENTAcyl coenzyme A acyltransferase-1 (Acat-1) is critical to lipid droplet formation, and thus inhibition of Acat-1 presents as a pharmacological target. Intratracheal administration of K-604, an Acat-1 inhibitor, reduces intracellular cholesterol ester accumulation in lung macrophages, attenuates inflammation and macrophage activation, and normalizes mediators of surface-active function after intratracheal bleomycin administration in a rodent model. The data presented within suggest that inhibition of Acat-1 in the lung improves acute lung injury outcomes.

Selonsertib Enhances Kidney Protection Beyond Standard of Care in a Hypertensive, Secondary Glomerulosclerosis CKD Model.

Despite widespread use of renin-aldosterone-angiotensin system inhibitors and the benefits of lowering glomerular pressure in patients with CKD, there remains a major unmet need for therapies targeting underlying causes of CKD progression. Apoptosis signal-regulating kinase 1 (ASK1) promotes apoptosis and glomerulosclerosis, and is implicated in the progression of diabetic kidney disease (DKD), a major cause of CKD. Selonsertib is a selective ASK1 inhibitor currently in clinical development for the treatment of DKD. We examined the added benefits of selonsertib on existing glomerulosclerosis and related molecular pathways in the nondiabetic 5/6 nephrectomy (5/6 Nx) rat model in combination with the angiotensin-converting enzyme inhibitor (ACEI) enalapril. Male Sprague Dawley rats underwent 5/6 Nx with kidney biopsy 8 weeks later for assessment of glomerulosclerosis, and were randomized to four treatment groups with equal glomerulosclerosis: selonsertib, enalapril, combination (selonsertib plus enalapril), and untreated controls. Serum creatinine, systolic BP (SBP), and urinary albumin were measured at intervals. Animals were euthanized at week 12 for histologic, biochemical, and molecular analyses. All rats developed hypertension, albuminuria, and glomerulosclerosis by week 8. Kidney function further declined, and glomerulosclerosis and albuminuria progressively increased in controls from week 8 to 12. Enalapril treatment alone from week 8 to 12 reduced SBP versus controls, decreased albuminuria, and resulted in numerically lower glomerulosclerosis. Selonsertib alone had no effect on SBP but preserved kidney function. Combined treatment significantly reduced glomerulosclerosis, with more regression than either monotherapy. Enalapril treatment resulted in fewer interstitial macrophages, whereas selonsertib treatment reduced apoptosis and podocyte loss. RNA-seq revealed that combined treatment influenced pathways related to extracellular matrix and wound healing. Selonsertib targets a novel, nonhemodynamic pathway in CKD. Our data suggest that ASK1 inhibition, when combined with ACEI, has additive effects to reduce progression of glomerulosclerosis, attenuate kidney function decline, and reduce podocyte loss.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Increased Heme Oxygenase 1 Expression upon a Primary Exposure to the Respiratory Syncytial Virus and a Secondary Mycobacterium bovis Infection.

The human respiratory syncytial virus (hRSV) is the leading cause of severe lower respiratory tract infections in infants. Because recurrent epidemics based on reinfection occur in children and adults, hRSV has gained interest as a potential primary pathogen favoring secondary opportunistic infections. Several infection models have shown different mechanisms by which hRSV promotes immunopathology to prevent the development of adaptive protective immunity. However, little is known about the long-lasting effects of viral infection on pulmonary immune surveillance mechanisms. As a first approach, here we evaluated whether a primary infection by hRSV, once resolved, dampens the host immune response to a secondary infection with an attenuated strain of Mycobacterium bovis (M. Bovis) strain referred as to Bacillus Calmette-Guerin (BCG). We analyzed leukocyte dynamics and immunomodulatory molecules in the lungs after eleven- and twenty-one-days post-infection with Mycobacterium, using previous hRSV infected mice, by flow cytometry and the expression of critical genes involved in the immune response by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Among the latter, we analyzed the expression of Heme Oxygenase (HO)-1 in an immunization scheme in mice. Our data suggest that a pre-infection with hRSV has a conditioning effect promoting lung pathology during a subsequent mycobacterial challenge, characterized by increased infiltration of innate immune cells, including interstitial and alveolar macrophages. Our data also suggest that hRSV impairs pulmonary immune responses, promoting secondary mycobacterial colonization and lung survival, which could be associated with an increase in the expression of HO-1. Additionally, BCG is a commonly used vaccine that can be used as a platform for the generation of new recombinant vaccines, such as a recombinant BCG strain expressing the nucleoprotein of hRSV (rBCG-N-hRSV). Therefore, we evaluated if the immunization with rBCG-N-hRSV could modulate the expression of HO-1. We found a differential expression pattern for HO-1, where a higher induction of HO-1 was detected on epithelial cells compared to dendritic cells during late infection times. This is the first study to demonstrate that infection with hRSV produces damage in the lung epithelium, promoting subsequent mycobacterial colonization, characterized by an increase in the neutrophils and alveolar macrophages recruitment. Moreover, we determined that immunization with rBCG-N-hRSV modulates differentially the expression of HO-1 on immune and epithelial cells, which could be involved in the repair of pulmonary tissue.

Susceptibility of renal fibrosis in diabetes: Role of hypoxia inducible factor-1.

Diabetes may prevent kidney repair and sensitize the kidney to fibrosis or scar formation. To test this possibility, we examined renal fibrosis induced by unilateral ureteral obstruction (UUO) in diabetic mouse models. Indeed, UUO induced significantly more renal fibrosis in both Akita and STZ-induced diabetic mice than in nondiabetic mice. The diabetic mice also had more apoptosis and interstitial macrophage infiltration during UUO. In vitro, hypoxia induced higher expression of the fibrosis marker protein fibronectin in high glucose-conditioned renal tubular cells than in normal glucose cells. Mechanistically, hypoxia induced significantly more hypoxia-inducible factor-1 α (HIF-1 α) in high glucose cells than in normal glucose cells. Inhibition of HIF-1 attenuated the expression of fibronectin induced by hypoxia in high-glucose cells. Consistently, UUO induced significantly higher HIF-1α expression along with fibrosis in diabetic mice kidneys than in nondiabetic kidneys. The increased expression of fibrosis induced by UUO in diabetic mice was diminished in proximal tubule-HIF-1α-knockout mice. Together, these results indicate that diabetes sensitizes kidney tissues and cells to fibrogenesis probably by enhancing HIF-1 activation.

Targeted nanotherapy with everolimus reduces inflammation and fibrosis in scleroderma-related interstitial lung disease developed by PSGL-1 deficient mice.

Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

ScRNA-seq expression of IFI27 and APOC2 identifies four alveolar macrophage superclusters in healthy BALF.

Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs, interstitial macrophages, and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified two MARCKS+LGMN+IMs, FOLR2+SELENOP+ and SPP1+PLA2G7+ IMs, monocyte subtypes, DC1, DC2, migDCs, plasmacytoid DCs, lymphocytes, epithelial cells, and four AM superclusters (families) based on the gene expression of IFI27 and APOC2 These four AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, cholesterol, chemokine, metallothionein, interferon, and small-cluster AMs. Interestingly, the chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Last, other AM datasets were projected onto our dataset. Similar AM superclusters and functional subclusters were observed, along with a significant increase in chemokine and IFN AM subclusters in individuals infected with COVID-19. Overall, functional specializations of the AM subclusters suggest that there are highly regulated AM niches with defined programming states, highlighting a clear division of labor.

Gasdermin D is involved in switching from apoptosis to pyroptosis in TLR4-mediated renal tubular epithelial cells injury in diabetic kidney disease

BackgroundToll-like receptor 4 (TLR4) mediated pathway plays a pivotal role in promoting tubulointerstitial inflammation and contributes to the progression in type 2 diabetic kidney disease (T2DKD). As the first identified key pyroptosis executor, gasdermin D (GSDMD) is activated by caspases and might be the key protein to switch apoptosis to pyroptosis. It remains unclear that role of TLR4 on canonical pyroptosis pathway, and whether GSDMD is involved in switching from apoptosis to pyroptosis in the TLR4-related tubular injury in T2DKD. MethodsImmunohistochemistry staining was used to detect the expression of pyroptosis-related proteins in renal tissues of T2DKD patients. T2DKD models was induced in TLR4 knockout (TLR4−/−) mice through a high-fat diet combined with streptozotocin. Pyroptosis (caspase-1, GSDMD, interleukin 18(IL-18), interleukin 1β(IL-1β)) and apoptosis levels (caspase-3, Bax and Bcl-2) were detected by Western blot. HK-2 cells were cultured under high-glucose (HG) conditions as an in vitro model and then challenged with a TLR4-specific antagonist (TAK-242). GSDMD small interfering RNA (siRNA) and overexpression plasmid were transfected into HK-2 cells to down- or up-regulate GSDMD. The pyroptosis and apoptosis rates were determined by flow cytometry. ResultsThe expression levels of caspase-1, GSDMD, IL-18 and IL-1β were increased in renal biopsy tissues of T2DKD patients and GSDMD expression was positively correlated with tubular injury. Silencing GSDMD attenuated HG-induced IL-18, IL-1β, FN and α-SMA, and reduced pyroptotic cells rate in HK-2 cells. Up-regulation of GSDMD inhibited HG-induced expression of Bax and cleaved caspase-3 and reduced apoptosis rate. TLR4 knockout alleviated tubular injury and interstitial macrophages infiltration, improved impaired renal dysfunction, and decreased the expressions of active N-terminal of GSDMD(GSDMD-N), cleaved caspase-1(cl-caspase-1) and cleaved caspase-3(cl-caspase-3) in T2DKD mice. TLR4 inhibition reduced HG-induced pyroptosis and apoptosis level in HK-2 cells, while GSDMD up-regulation increased pyroptosis rate and decreased apoptosis rate. ConclusionsTLR4 could exacerbate tubular injury and fibrosis via GSDMD-mediated canonical pyroptosis pathway in T2DKD. Activation of GSDMD could inhibit apoptosis and activate pyroptosis, which may involve the potential switch mechanism between TLR4-mediated pyroptosis and apoptosis in T2DKD.

Effects of NLRP3/Caspase-1 pathway on Banxia Houpu decoction in the intervention of chronic intermittent hypoxia in mice with renal inflammatory injury

Objective: To investigate the effects of Banxia Houpo decoction on the renal NLRP3/Caspase-1/IL-1β signaling pathway in chronic intermittent hypoxia mice. Methods: C57BL/6 mice were randomly divided into 3 groups, normal control group (Control), chronic intermittent hypoxia group (CIH), and Banxia Houpo decoction treatment group (BHD), with 10 mice in each group. Mice in the CIH group and BHD group were placed in a hypoxic chamber. The oxygen volume fraction in the cabin was decreased from 21% to 9% in 90 s, and then oxygen was filled in 90 s to gradually increase the oxygen volume fraction in the cabin to 21%, while the mice in the control group were placed in the cabin and filled with normal air, processing 8 hours per day for 21 days. The mice in BHD group were treated with Banxia Houpu decoction by gavage before entering the cabin every day, and the control group and CIH group were given an equal volume of normal saline. After modeling, the changes of renal function indexes in each group were detected; HE and Masson staining were used to observe the pathological conditions of the kidney; Western blot and immunohistochemical staining were used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), aspartate-specific cysteine protein 1(Caspase-1) and interleukine-1beta(IL-1β). Results: Compared with control group, the contents of serum renal functional indexes UA, BUN and SCr in CIH group were increased significantly (P<0.01), and after BHD treatment, they all were decreased significantly compared with CIH group (P<0.01). Compared with control group, the results of HE staining showed that in the CIH group, glomerular endothelial cells were degenerated and necrotic, and vacuoles of different sizes appeared in renal tubular epithelial cells, and a small amount of renal tubular epithelial cells fell off and died. The pathological condition of the BHD group was improved compared with CIH group, the glomerular morphology gradually returned to normal, and a small amount of renal tubular epithelial cells fell off and died. Compared with control group, Masson staining results showed that there was obvious fibrosis around the glomeruli in the CIH group, the fibrosis was significantly reduced in the BHD group. The expression levels of NLRP3, Caspase-1, IL-1β and IL-18 were increased significantly compared with control group (P<0.05 or P<0.01), and immunohistochemical staining showed that NLRP3 was mainly expressed in renal tubular epithelial cells and interstitial macrophages, caspase-1 and IL-1β were mainly found in the cytoplasm of renal tubular epithelial cells. After BHD treatment, the expression levels of each protein were decreased compared with CIH group (P<0.05). Conclusion: Banxia Houpu decoction can reduce the kidney damage by inhibiting the expression of related molecules in the NLRP3/Casapse-1/IL-1β signaling pathway.

Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c− interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r−/− mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.

Abstract 1307: Modulation of the immune environment in a preclinical orthotopic model of non-small cell lung cancer exposed to ablative doses of radiation

Abstract Stereotactic body radiation therapy (SBRT) allows the administration of ablative doses and is used for the management of a proportion of non-small cell lung cancer (NSCLC) patients. SBRT allows high fractional doses (≥ 8 Gy), which may result in a fundamentally different biological effect from conventional radiotherapy (RT). The effects of SBRT are mediated by cytotoxic, inflammatory and immunomodulatory mechanisms. The latter are of particular interest and their understanding is the key to potentiating the local effect and synergy with immunotherapies. Several studies have shown the involvement of the immune system in the antitumor efficacy of RT, but these studies were mainly conducted in subcutaneous murine tumor models using conventional doses of RT. In contrast, the effects of ablative irradiation (IR) doses on the infiltration and activity of immune cells are poorly understood, especially in orthotopic models.Here we used a murine orthotopic model of NSCLC to evaluate the effects of high doses of RT, delivered using a small animal radiation platform (SARRP) that allows stereotactic IR with a millimetric precision. Mice were subjected to a trans-pleural injection with Lewis lung carcinoma cells expressing luciferase (LLC-Luc) and irradiated at doses ranging from 8Gy to 60Gy in 1-3 fractions. Our results show that orthotopic LLC tumors, which correctly develop as single tumor mass within the lung parenchyma, are resistant to SBRT, with no complete tumor regression observed even after 60Gy IR. The characterization of the tumor environment by at the clinically relevant dose of 20Gy showed transient (by day 3 post-IR) increased CD8+ T cells and inflammatory monocytes within the tumor bed but not in the juxtatumor tissue. Resident alveolar macrophages where mostly detected in the juxtatumor tissue and were depleted by RT. Although RT did not affect interstitial tumor-associated macrophages (TAM) number, we observed that the fraction of CD206+ TAM was decreased together with an increased proportion of MHC class II+ TAM. At later time points, Treg harboring increased CD69 accumulated in the tumor bed and TAM recovered a similar phenotype compared to non-irradiated mice. The analysis of the tumor cytokinome revealed the modulation of several cytokines and chemokines including CCL-5, CXCL-10, LIF (Leukemia Inhibitory Factor) and Eotaxin. Altogether, these results argue that high IR doses induce transient and localized activation of the tumor immune environment that is rapidly dampened by anti-inflammatory cell recovery, such as Tregs and CD206 TAMs. The ongoing analyses using a single-cell RNAseq approach on sorted CD45+ cells will allow a precise evaluation of the composition and phenotype of the tumor immune populations.The results of this project will guide the design of therapeutic combinations with immunomodulatory agents to optimize the tumor response to SBRT. Citation Format: Céline Clémenson, Morgane Dos Santos, Maxime Petit, Jeremy Lavigne, Winchygn Liu, Marina Milic, Georges Tarlet, Agnès François, Alexandre Boissonnas, Fabien Milliat, Michele Mondini, Eric Deutsch. Modulation of the immune environment in a preclinical orthotopic model of non-small cell lung cancer exposed to ablative doses of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1307.