Neutrophil-attracting Chemokines Research Articles

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.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd−/−) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1β and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd−/− mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

DOCK8 is essential for neutrophil mediated clearance of cutaneous S. aureus infection

DOCK8 deficient patients are susceptible to skin infection with Staphylococcus aureus which is normally cleared by neutrophils. We examined the mechanism of this susceptibility in mice. Dock8−/− mice had delayed clearance of S. aureus from skin mechanically injured by tape stripping. The numbers and viability of neutrophils in infected but not in uninfected, tape stripped skin were significantly reduced in Dock8−/− mice compared to WT controls. This is despite comparable numbers of circulating neutrophils, and normal to elevated cutaneous expression of Il17a and IL-17A inducible neutrophil attracting chemokines Cxcl1, Cxcl2 and Cxcl3. DOCK8 deficient neutrophils were significantly more susceptible to cell death upon in vitro exposure to S. aureus and exhibited reduced phagocytosis of S. aureus bioparticles but had a normal respiratory burst. Impaired neutrophil survival in infected skin and defective neutrophil phagocytosis likely underlie the susceptibility to cutaneous S. aureus infection in DOCK8 deficiency.

Disease associated monocytes contribute to the inflammatory milieu in hidradenitis suppurativa and have dysregulated expression of transposable elements

Abstract Hidradenitis suppurativa (HS), an inflammatory skin-disease, manifests as painful lesions in intertriginous skin. Despite their central role in innate immunity the role of monocytes in HS is largely unknown. Here we utilized cytometry by time-of-flight (CyTOF), imaging CyTOF (IMC), cellular indexing of transcriptomes and epitopes by sequencing (CITEseq), spatial transcriptomics (ST), and our recently developed database (HSOmicsDB) to characterize the systemic and cutaneous immune response in patients with HS. CITEseq found differential genes in circulating immune cells from HS patients enriched for cellular activation and extravasation. CyTOF immune profiling of fresh whole-blood samples found decreased frequency of classical Mos (C.Mos), which had increased expression of skin homing chemokine receptors. IMC found C.Mos infiltrated lesional dermis and tunnel regions, but not perilesional (PL) skin. Patients with HS showed increased proportions of activated C.Mo subclusters and a population of SOD2+ C.Mos expressing genes downstream of viral sensing pathways. SOD2 +C.Mos expressed high levels of neutrophil attracting chemokines. ST found SOD2 +C.Mos localized to regions of active inflammation and dermal tunnels. HSOmicsDB showed Mos and Macs expressed SOD2 +C.Mo genes, which were enriched in lesions, but not PL skin. Finally, we show that SOD2 +C.mos in HS have dysregulated expression of transposable elements (TEs), which may be activating viral sensing mechanisms in patients with HS. Together our data shows monocytes in HS function to recruit neutrophils to lesional HS skin, and provides the first evidence implicating TEs as disease promoting factors in HS by leading to C.Mo dysregulation. Supported By NIH/NIAMS, R01AR078688-01A1 and R21AR079089-01A1

Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth

BackgroundInflammation-related predisposition to cancer plays an essential role in cancer progression and is associated with poor prognosis. A hypoxic microenvironment and neutrophil infiltration are commonly present in solid tumours, including gastric cancer (GC). Neutrophil extracellular traps (NETs) have also been demonstrated in the tumour immune microenvironment (TIME), but how NETs affect GC progression remains unknown. Here, we investigated the role of NET formation in the TIME and further explored the underlying mechanism of NETs in GC tumour growth.MethodsHypoxia-induced factor-1α (HIF-1α), citrulline histone 3 (citH3) and CD66b expression in tumour and adjacent nontumor tissue samples was evaluated by western blotting, immunofluorescence and immunohistochemical staining. The expression of neutrophil-attracting chemokines in GC cells and their hypoxic-CM was measured by qRT‒PCR and ELISA. Neutrophil migration under hypoxic conditions was evaluated by a Transwell assay. Pathway activation in neutrophils in a hypoxic microenvironment were analysed by western blotting. NET formation was measured in vitro by immunofluorescence staining. The protumour effect of NETs on GC cells was identified by Transwell, wound healing and cell proliferation assays. In vivo, an lipopolysaccharide (LPS)-induced NET model and subcutaneous tumour model were established in BALB/c nude mice to explore the mechanism of NETs in tumour growth.ResultsGC generates a hypoxic microenvironment that recruits neutrophils and induces NET formation. High mobility group box 1 (HMGB1) was translocated to the cytoplasm from the nucleus of GC cells in the hypoxic microenvironment and mediated the formation of NETs via the toll-like receptor 4 (TLR4)/p38 MAPK signalling pathway in neutrophils. HMGB1/TLR4/p38 MAPK pathway inhibition abrogated hypoxia-induced neutrophil activation and NET formation. NETs directly induced GC cell invasion and migration but not proliferation and accelerated the augmentation of GC growth by increasing angiogenesis. This rapid tumour growth was abolished by treatment with the NET inhibitor deoxyribonuclease I (DNase I) or a p38 MAPK signalling pathway inhibitor.ConclusionsHypoxia triggers an inflammatory response and NET formation in the GC TIME to augment tumour growth. Targeting NETs with DNase I or HMGB1/TLR4/p38 MAPK pathway inhibitors is a potential therapeutic strategy to inhibit GC progression.Ca2HLG-e4J4ceVPYPV7JaoVideo

Trajectory of neutrophilic responses in a mouse model of pollutant-aggravated allergic asthma

Experimental studies suggest that neutrophils could contribute to allergic asthma pathogenesis, that is mainly driven by type 2 immunity. Inhalation of diesel exhaust particles (DEP) is implicated in both exacerbation and development of asthma. Since exposure to DEP is associated with a neutrophilic component, we aimed to investigate how exposure to the combination of allergens and DEP modulates neutrophilic responses. Human bronchial epithelial cells (HBEC) were exposed to house dust mite (HDM), DEP or HDM + DEP in vitro to determine the expression of neutrophil-recruiting chemokines. Female (C57BL/6 J) mice were intranasally instilled with saline, DEP, HDM or combined HDM + DEP for 3 weeks (subacute) or 6 weeks (chronic). The neutrophilic responses were determined in lung tissue and bronchoalveolar lavage fluid (BALF). Simultaneous exposure to HDM + DEP resulted in increased CXCL1 and CXCL8 mRNA expression by HBEC in vitro. In mice, subacute exposure to HDM + DEP induced a strong mixed eosinophilic/neutrophilic inflammation in BALF and lung and was associated with higher expression of neutrophil-attracting chemokines and NET formation compared to the sole exposures. After chronic HDM + DEP exposure, a similar neutrophilic response was observed, however the NET formation was less pronounced. Interestingly, the increase of BALF eosinophils was also significantly attenuated after chronic HDM + DEP exposure compared to the subacute exposure. Subacute and chronic HDM + DEP exposure induced goblet cell hyperplasia and airway hyperresponsiveness. Our data suggest a role for neutrophils and NETs in pollutant-aggravated eosinophilic allergic asthma. Moreover, subacute exposure to HDM + DEP induces a mixed eosinophilic/neutrophilic response whereas upon chronic HDM + DEP exposure there is a shift in inflammatory response with a more prominent neutrophilic component.

Staphylococcus aureus Infection Induces the Production of the Neutrophil Chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by Murine Osteoblasts.

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

P068 Polyunsaturated fatty acid excess fuels pro-inflammatory transcriptional networks in GPX4 deficient intestinal epithelial cells

Abstract Background There is increasing evidence that the rise in Crohn’s disease (CD) incidence is partially driven by constituents of a Western-style diet such as polyunsaturated fatty acids (PUFA). Recently we could demonstrate that PUFA excess induces a CD-like enteritis in mice lacking antioxidant GPX4 in their intestinal epithelial cells (IECs) and that CD patients clinical disease activity positively correlates with PUFA intake. In this project we investigated the role of epithelial lipid composition and lipid mediators during PUFA induced metabolic gut inflammation. Furthermore, we transcriptionally profiled GPX4 deficient small intestinal cells after stimulation with ω3- and ω6-PUFA. Methods Mice lacking one allele of Gpx4 in their IECs (GPX4+/-IEC) and wild-type controls (WT) were fed a PUFA (10% fish oil) enriched Western diet (PUFA-WD) for 12 weeks. Intestinal inflammation was assessed by histopathology. IECs were harvested by scraping of small intestinal sections and lipid profiles and mediators were analysed with LC/MS. GPX4 in murine small intestinal cells (MODE-K) was knocked down by siRNA (siGpx4) scrambled siRNA served as control (siCtrl). MODE-K were stimulated with ω3-, ω6-PUFA, 5,6- and 8,9-DHET or vehicle. Cytokine expression in cell culture supernatant was analysed by ELISA and transcriptome was analysed by bulk RNA sequencing. Results Histopathology revealed gut inflammation in GPX4+/-IEC but not WT mice after 12 weeks PUFA-WD feeding. LC/MS analysis of IECs derived from GPX4+/-IEC and WT mice fed the PUFA-WD depicted a significant increase in eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6) containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species (Fig 1a). All other PE and PC species showed a reduced amount, confirming the enrichment of the specific PUFAs within the IECs by the PUFA-WD. Lipid mediator analysis further revealed an increase in 5,6-DHET and 8,9-DHET only in IECs derived from GPX4+/-IEC but not WT mice (Fig 1b). When stimulated with PUFA, siGpx4 MODE-K produced high amounts of neutrophil attracting chemokine CXCL-1 which is attenuated by the supplementation of anti-inflammatory lipid mediators 5,6-DHET and 8,9-DHET (Fig 2). Transcriptome analysis revealed the upregulation of pro-inflammatory pathways including TNFa, MAPK and NF-κB in siGpx4 vs. siCtrl MODE-K after PUFA stimulation (Fig 3). Conclusion By integrating lipidomic and transcriptomic data of a reductionist cell culture system and a model organism, we could show that PUFA excess in a GPX4 deficient individual fuels a pro-inflammatory transcriptional network which outweighs anti-inflammatory ω3-PUFA metabolites influence culminating in gut inflammation.

Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

Interleukin-19 Levels Are Increased in Palmoplantar Pustulosis and Reduced following Apremilast Treatment.

Palmoplantar pustulosis (PPP) is a chronic inflammatory skin disease characterised by neutrophilic granulocyte (neutrophil)-filled pustules on the palms and soles. The pathogenesis of PPP is poorly understood. This study conducted an identification of the immune mediators associated with PPP and an exploration of apremilast treatment effects on them. We screened for immune mediators elevated in blood taken from 68 patients with PPP versus control participants and included the most promising parameters in the protocol of phase the 2, multicentre study of apremilast (PDE4 inhibitor) in 21 patients with moderate-to-severe PPP (APLANTUS; EudraCT 2016-005122-11) for respective analysis of blood and skin samples of study patients. We investigated stimulated neutrophils and three-dimensional reconstituted epidermis cultures. Interleukin (IL)-19 was found to be the most upregulated immune mediator in the blood of PPP patients. IL-19 serum levels were independent of patients' age, gender, and BMI but were associated with strongly upregulated cutaneous IL-19 expression and correlated with the number of palmoplantar pustules. In patients participating in the APLANTUS study, apremilast reduced pustules more effectively than erythema and scaling. Moreover, this treatment significantly reduced IL-19 blood and skin levels. The reduction in IL-19 blood levels at week 4 correlated with the reduction in pustule counts at week 20 (end of treatment). IL-19 was expressed by neutrophils activated in vitro and induced CXCL6, a neutrophil-attracting chemokine, in epidermis models. This work demonstrates elevated IL-19 levels in the blood and skin of PPP patients and suggests a relevant role of this cytokine in the appearance of pustules in this disorder. It also suggests the suitability of IL-19 blood levels as a predictive biomarker for the treatment response of PPP patients, which should be validated in further studies.

TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.

824 Spatial transcriptomic analysis of HS skin lesions reveals that tunnels are immunologically active and activity correlates with disease severity

Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by neutrophil-rich inflammatory nodules, abscesses or tunnels (sinus tracts). Tunnels, unique to HS, are deep dermal cavities lined with squamous epithelium that are immunologically active, contributing to disease pathology. The role of tunnels in driving ongoing disease is poorly understood. We used the GeoMX Digital Spatial Profiler to analyze a moderate (Hurley stage II) compared to a severe (Hurley stage III) HS skin lesion to explore biological differences between and within lesions. Four distinct regions per sample were analyzed: tunnel epithelium, immune infiltrate adjacent to a tunnel, immune infiltrate not associated with a tunnel, and sub-epidermis. We observed higher expression of keratinization genes (e.g. K16, S100A7) in the more differentiated tunnel epithelium of the severe lesion compared to the moderate lesion tunnel. Genes for proinflammatory cytokines (e.g. IL36, IL1B), chemokines (e.g. CXCL1, CCL20), neutrophil activation (e.g. ELANE, MPO) and B cells (CD79A, SDC1) had higher expression in the severe compared to moderate HS lesion. We found a spatial gradient in gene expression for IL36 and neutrophil-attracting chemokines (CXCL1, CXCL8), from high expression in the tunnel epithelium to lower expression in the sub-epidermis. Both lesions displayed these gradients, but those in the severe HS lesion were more pronounced. Conversely, spatial gradients of fibroblast-related genes (e.g. PDGFR, FAP) had higher expression in the sub-epidermis and lower expression adjacent to tunnel epithelium. These data suggest a marked increase in dermal inflammation in more severe HS lesions. The spatial gradients of cytokine and chemokine gene expression support the idea that more differentiated tunnels are more immunologically active, potentially contributing to ongoing severe disease, rather than representing remnants of prior disease.

CD11b suppresses TLR activation of nonclassical monocytes to reduce primary graft dysfunction after lung transplantation.

Primary graft dysfunction (PGD) is the leading cause of postoperative mortality in lung transplant recipients and the most important risk factor for development of chronic lung allograft dysfunction. The mechanistic basis for the variability in the incidence and severity of PGD between lung transplant recipients is not known. Using a murine orthotopic vascularized lung transplant model, we found that redundant activation of Toll-like receptors 2 and 4 (TLR2 and -4) on nonclassical monocytes activates MyD88, inducing the release of the neutrophil attractant chemokine CXCL2. Deletion of Itgam (encodes CD11b) in nonclassical monocytes enhanced their production of CXCL2 and worsened PGD, while a CD11b agonist, leukadherin-1, administered only to the donor lung prior to lung transplantation, abrogated CXCL2 production and PGD. The damage-associated molecular pattern molecule HMGB1 was increased in peripheral blood samples from patients undergoing lung transplantation after reperfusion and induced CXCL2 production in nonclassical monocytes via TLR4/MyD88. An inhibitor of HMGB1 administered to the donor and recipient prior to lung transplantation attenuated PGD. Our findings suggest that CD11b acts as a molecular brake to prevent neutrophil recruitment by nonclassical monocytes following lung transplantation, revealing an attractive therapeutic target in the donor lung to prevent PGD in lung transplant recipients.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On theone hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Neutrophil dynamics in the tumor microenvironment determines therapy efficacy and is regulated by microbiota

Abstract Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome. Supported by NIH Intramural Program

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.

Basophil-derived IL-4 promotes cutaneous Staphylococcus aureus infection.

Superficial cutaneous Staphylococcus aureus (S. aureus) infection in humans can lead to soft tissue infection, an important cause of morbidity and mortality. IL-17A production by skin TCRγδ+ cells in response to IL-1 and IL-23 produced by epithelial and immune cells is important for restraining S. aureus skin infection. How S. aureus evades this cutaneous innate immune response to establish infection is not clear. Here we show that mechanical injury of mouse skin by tape stripping predisposed mice to superficial skin infection with S. aureus. Topical application of S. aureus to tape-stripped skin caused cutaneous influx of basophils and increased Il4 expression. This basophil-derived IL-4 inhibited cutaneous IL-17A production by TCRγδ+ cells and promoted S. aureus infection of tape-stripped skin. We demonstrate that IL-4 acted on multiple checkpoints that suppress the cutaneous IL-17A response. It reduced Il1 and Il23 expression by keratinocytes, inhibited IL-1+IL-23–driven IL-17A production by TCRγδ+ cells, and impaired IL-17A–driven induction of neutrophil-attracting chemokines by keratinocytes. IL-4 receptor blockade is shown to promote Il17a expression and enhance bacterial clearance in tape-stripped mouse skin exposed to S. aureus, suggesting that it could serve as a therapeutic approach to prevent skin and soft tissue infection.

Epidermis-Intrinsic Transcription Factor Ovol1 Coordinately Regulates Barrier Maintenance and Neutrophil Accumulation in Psoriasis-Like Inflammation

Skin epidermis constitutes the exterior barrier that protects the body from dehydration and environmental assaults. Barrier defects underlie common inflammatory skin diseases, but the molecular mechanisms that maintain barrier integrity and regulate epidermal-immune cell cross-talk in inflamed skin are not fully understood. In this study, we show that skin epithelia-specific deletion of Ovol1, which encodes a skin disease‒linked transcriptional repressor, impairs the epidermal barrier and aggravates psoriasis-like skin inflammation in mice in part by enhancing neutrophil accumulation and abscess formation. Through molecular studies, we identify IL-33, a cytokine with known pro-inflammatory and anti-inflammatory activities, and Cxcl1, a neutrophil-attracting chemokine, as potential weak and strong direct targets of Ovol1, respectively. Furthermore, we provide functional evidence that elevated Il33 expression reduces disease severity in imiquimod-treated Ovol1-deficient mice, whereas persistent accumulation and epidermal migration of neutrophils exacerbate it. Collectively, our study uncovers the importance of an epidermally expressed transcription factor that regulates both the integrity of the epidermal barrier and the behavior of neutrophils in psoriasis-like inflammation.

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome

Enterohaemorrhagic E.coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E.coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E.coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E.coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.