anti-Ly6G Antibody Research Articles

Caloric Restriction Preserves BBB Integrity After Transient Focal Cerebral Ischemia Through Reducing Neutrophil Infiltration.

Caloric restriction is a health-promoting lifestyle that has been reported to protect both white and gray matter in cases of ischemic stroke. This study will explore the underlying mechanism of restricted feeding (RF) and provide a theoretical basis for precise clinical treatment of stroke. In this study, we pretreated C57BL/6J mice with 70% RF for a continuous 28-day period prior to 60 min of transient focal cerebral ischemia (tFCI). Histological staining, diffusion tensor imaging (DTI), and behavioral assessments were used to assess RF's neuroprotection following tFCI. Immunofluorescence staining, quantitative real-time PCR, and flow cytometry were conducted to evaluate brain inflammation post-tFCI. Western blot, immunofluorescence staining, tracers, and electric microscopy were used to assess the blood-brain barrier (BBB) integrity. Peripheral neutrophils were cleared by administrating an anti-Ly6G antibody. Initially, DTI, NeuN staining, and a batch of behavioral tests verified that RF significantly mitigated both gray/white matter injury and neurological deficits in the short- and long-term following tFCI. RF mice showed more anti-inflammatory microglia in their brains, along with reduced inflammatory cytokines, and chemokines. Interestingly, RF significantly reduced the neutrophils and macrophage infiltration. Subsequently, we observed that RF mice exhibited better BBB integrity following tFCI, with reduced neutrophil infiltration and matrix metalloprotein-9 release. Furthermore, the clearance of neutrophils with anti-Ly6G antibody in adlibitum feeding mice (LF-Ly6G) elicited comparable neuroprotective effects to those observed in RF, including improvements in neurological deficits, reductions in infarct volume, and mitigation of BBB damage. In summary, our findings suggest that RF maintains the BBB integrity following ischemic stroke at least partially by reducing neutrophil infiltration, thereby alleviating both neurological and histological impairments.

PMN-MDSCs are responsible for immune suppression in anti-PD-1 treated TAP1 defective melanoma.

The transporter associated with antigen processing (TAP) is a key component of the classical HLA I antigen presentation pathway. Our previous studies have demonstrated that the downregulation of TAP1 contributes to tumor progression and is associated with an increased presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. However, it remains unclear whether the elevation of MDSCs leads to immune cell exhaustion in tumors lacking TAP1. In this study, we established mouse models of tumors with TAP1 deficiency, and we employed PMN-MDSC depletion to investigate their impact on the immune microenvironment within the tumors. We found that MDSC depletion significantly altered the immune-suppressive effects of TAP1-deficient tumor when anti-PD-1 treatment was administered. Targeting PMN-MDSC may be a promising therapeutic strategy for the treatment of tumors with TAP1 deficiency during ICB treatment. Immunohistochemistry (IHC) was conducted to assess TAP1 expression in mouse melanoma tissues. Ly6G, F4/80, and NKp46 markers were detected in B16 parental and TAP1 knockout tissues, respectively. To enhance anti-tumor immunity, hyperthermia-treated B16F10 WT cell suspension was injected prior to tumor cell introduction. Subsequently, we established B16F10 TAP1 knockout and WT melanoma mouse models. Tumors were collected, and the immune microenvironment was monitored accordingly. Anti-Ly6G antibody was administered to deplete polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Finally, flow cytometry analysis for immune infiltration, quantitative PCR for cytokine levels, and immunofluorescence assays were performed to analyze the immune response. The level of Ly6G+ cell infiltration was significantly higher in samples exhibiting low TAP1 expression, while no differences were observed in the infiltration of F4/80+ cells or NKp46+ cells. Furthermore, the immune-suppressive effects associated with PMN-MDSCs were reversed following their elimination; this resulted in an increase in CD8+ T cells and a higher ratio of CD8+ T cells to Tregs, while the infiltration of innate immune cells remained unaffected. Functional markers of these immune cells indicated an active anti-tumoral immune response following the removal of PMN-MDSCs. Quantitative PCR analysis indicated elevated levels of TNF-α and IL-6, accompanied by decreased levels of TGF-β in the tumor microenvironment of TAP1. Our data indicate that myeloid-derived suppressor cells (PMN-MDSCs) play an essential role in creating a tumorigenic immune microenvironment in TAP1 knockout tumors. Therefore, targeting PMN-MDSCs may become a promising therapeutic strategy for the treatment of tumors with TAP1 deficiency during ICB treatment.

NET formation-mediated in situ protein delivery to the inflamed central nervous system

Delivering protein drugs to the central nervous system (CNS) is challenging due to the blood-brain and blood-spinal cord barrier. Here we show that neutrophils, which naturally migrate through these barriers to inflamed CNS sites and release neutrophil extracellular traps (NETs), can be leveraged for therapeutic delivery. Tannic acid nanoparticles tethered with anti-Ly6G antibody and interferon-β (aLy6G-IFNβ@TLP) are constructed for targeted neutrophil delivery. These nanoparticles protect interferon-β from reactive oxygen species and preferentially accumulate in neutrophils over other immune cells. Upon encountering inflammation, neutrophils release the nanoparticles during NET formation. In the female mouse model of experimental autoimmune encephalomyelitis, intravenous administration of aLy6G-IFNβ@TLP reduce disease progression and restore motor function. Although this study focuses on IFNβ and autoimmune encephalomyelitis, the concept of hitchhiking neutrophils for CNS delivery and employing NET formation for inflamed site-specific nanoparticle release can be further applied for delivery of other protein drugs in the treatment of neurodegenerative diseases.

Neutrophil swarming is crucial for limiting oral mucosal infection by Candida albicans.

Oral mucosal colonization by C. albicans (Ca) is benign in healthy people but progresses to deeper infection known as oropharyngeal candidiasis (OPC) that may become disseminated when combined with immunosuppression. Cortisone use and neutropenia are risk factors for invasive mucosal fungal infections, however the mechanisms are poorly understood. Here we identify in vivo neutrophil functional complexes known as swarms that are crucial for preventing Ca epithelial invasion. Anti-Ly6G antibody treatment impaired swarm formation and increased fungal infection depth confirming the role of neutrophil swarms in limiting Ca invasion. Neutrophil swarm function could be disrupted by administration of resolvins, and required leukotriene B4 receptor 1 (BLT1) expression so that administration of a leukotriene synthesis inhibitor reduced neutrophil swarm size permitting Ca invasion beyond the basement membrane. Cortisone treatment similarly reduced neutrophil swarming behavior and BLT1 expression and delayed expression of epithelial cytokines and chemokines. Thus, swarm structures have an important function in preventing deep invasion by C. albicans within the oral mucosa and represent a mechanism for increased disease severity under immune deficient clinical settings.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.

Cholestatic insult triggers alcohol-associated hepatitis in mice.

Alcohol-associated hepatitis (AH) is a severe, potentially life-threatening form of alcohol-associated liver disease with limited therapeutic options. Existing evidence shows that biliary dysfunction and cholestasis are common in patients with AH and are associated with poorer prognosis. However, the role of cholestasis in the development of AH is largely unknown. We aimed to examine the hypothesis that cholestasis can be an important etiology factor for AH. To study the interaction of cholestasis and alcohol, chronically ethanol (EtOH)-fed mice were challenged with a subtoxic dose of α-naphthylisothiocyanate (ANIT), a well-studied intrahepatic cholestasis inducer. Liver injury was measured by biochemical and histological methods. RNAseq was performed to determine hepatic transcriptomic changes. The impact of inflammation was assessed using an anti-LY6G antibody to deplete the neutrophils and DNase I to degrade neutrophil extracellular traps. ANIT synergistically enhanced liver injury following a 4-week EtOH feeding with typical features of AH, including increased serum levels of ALT, AST, and total bile acids, cholestasis, necrosis, neutrophil infiltration, and accumulation of neutrophil extracellular traps. RNAseq revealed multiple genes uniquely altered in the livers of EtOH/ANIT-treated mice. Analysis of differentially expressed genes suggested an enrichment of genes related to inflammatory response. Anti-LY6G antibody or DNase I treatment significantly inhibited liver damage in EtOH/ANIT-treated mice. Our results support the hypothesis that cholestasis can be a critical contributor to the pathogenesis of AH. A combined treatment of EtOH and ANIT in mice presents biochemical, histological, and molecular features similar to those found in patients with AH, suggesting that this treatment scheme can be a useful model for studying Alcohol-associated Cholestasis and Hepatitis (AlChoHep).

The role of neutrophils in pain: systematic review and meta-analysis of animal studies.

The peripheral inflammatory response is an attractive therapeutic target for pain treatment. Neutrophils are the first circulating inflammatory cells recruited to sites of injury, but their contribution to pain outcomes is unclear. We performed a systematic review and meta-analysis of original preclinical studies, which evaluated the effect of preemptive neutrophil depletion on pain outcomes (PROSPERO registration number: CRD42022364004). Literature search (PubMed, January 19, 2023) identified 49 articles, which were meta-analyzed using a random-effects model. The risk of bias was evaluated using SYRCLE's tool. The pooled effect considering all studies showed that neutrophil depletion induced a consistent pain reduction. Inflammatory, joint, neuropathic, and visceral pain showed significant pain alleviation by neutrophil depletion with medium-large effect sizes. However, muscle and postoperative pain were not significantly alleviated by neutrophil depletion. Further analysis showed a differential contribution of neutrophils to pain outcomes. Neutrophils had a higher impact on mechanical hyperalgesia, followed by nociceptive behaviors and mechanical allodynia, with a smaller contribution to thermal hyperalgesia. Interspecies (mice or rats) differences were not appreciated. Analyses regarding intervention unveiled a lower pain reduction for some commonly used methods for neutrophil depletion, such as injection of antineutrophil serum or an anti-Gr-1 antibody, than for other agents such as administration of an anti-Ly6G antibody, fucoidan, vinblastine, CXCR1/2 inhibitors, and etanercept. In conclusion, the contribution of neutrophils to pain depends on pain etiology (experimental model), pain outcome, and the neutrophil depletion strategy. Further research is needed to improve our understanding on the mechanisms of these differences.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis

ObjectiveAlcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges...

Hyperalgesic Effect Evoked by il-16 and its Participation in Inflammatory Hypernociception in Mice

The systemic administration of interleukin-16 (IL-16, 3–30 ng/kg) induced thermal hyperalgesia in mice, that was prevented by the acute injection of an anti-CD4 antibody (1 µg/kg), the depletion of circulating white blood cells by cyclophosphamide or the specific reduction of circulating CD4+ cells provoked by a high dose of an anti-CD4 antibody (30 µg/mouse, 24 h before). IL-16-induced hyperalgesia was locally inhibited after intraplantar (i.pl.) administration of the non-selective cyclooxygenase (COX) inhibitor diclofenac, the COX-1 inhibitor SC-560, the COX-2 inhibitor celecoxib, the TRPV1 antagonist capsazepine or the TRPA1 antagonist HC030031, thus demonstrating that prostaglandins and TRP channels are involved in this effect. The i.pl. administration of low doses of IL-16 (0.1–1 ng) evoked local hyperalgesia suggesting the possibility that IL-16 could participate in hypernociception associated to local tissue injury. Accordingly, IL-16 concentration measured by ELISA was increased in paws acutely inflamed with carrageenan or chronically inflamed with complete Freund´s adjuvant (CFA). This augmentation was reduced after white cell depletion with cyclophosphamide or neutrophil depletion with an anti-Ly6G antibody. Immunofluorescence and flow cytometry experiments showed that the increased concentration of IL-16 levels found in acutely inflamed paws is mainly related to the infiltration of IL-16+ neutrophils, although a reduced number of IL-16+ lymphocytes was also detected in paws inflamed with CFA. Supporting the functional role of IL-16 in inflammatory hypernociception, the administration of an anti-IL-16 antibody dose-dependently reduced carrageenan- and CFA-induced thermal hyperalgesia and mechanical allodynia. The interest of IL-16 as a target to counteract inflammatory pain is suggested.Graphical

Abstract We069: Post-infarction Cardiac Function is Improved in Mice with TSC2 -/- Macrophages

Introduction: Macrophages (MF) play major roles in myocardial ischemia/reperfusion (I/R) injury, both beneficial and detrimental. The mechanistic target of rapamycin (mTOR) plays an important role in MF polarization and functionality. It is prominently regulated by tuberous sclerosis complex 2 (TSC2) which constitutively inhibits and regulates mTORC1. Little is known about how such mTORC1 regulation influences the effect of MFs in I/R injured hearts. Hypothesis: We tested if gene knockdown of TSC2 to constitutively enhance MF mTORC1 activity by TSC2 knockdown impairs or benefits the I/R heart. Methods/Results: MF-specific TSC2 knock-out mice (MF TSC2-/- ) were generated by crossing flox’d TSC2 mice with those expressing LysM-Cre. MTORC1 was constitutively active in bone marrow-derived MF TSC2-/- , reflected by increased S6K1 and 4E-BP1 phosphorylation. MF TSC2-/- exposed to liposaccharide had greater proinflammatory responses, e.g. increased TNF-a secretion, supporting an M1-like phenotype. MF TSC2-/- also exhibited less M2-like features (reduced Arg1, Mrc1, and Rentla expression). To test the impact of MF TSC2-/- on infiltrating myocardial MF subtypes post I/R, MF were isolated from the myocardium and sorted by flow-cytometry based on CD45 + , CD11b + , and CD64 + and then parsed as ±CCR2 and ±MHC-II. We found MF TSC2-/- resulted in an increase in infiltrating CCR2 - MHC-II - phenotypes, which are thought to be anti-inflammatory and to support healing. Consistent with this, we found MF TSC2-/- hearts exposed to I/R had higher ejection fractions (74.2±10.3%) vs controls (47.2±14.3%, P=0.002). MF TSC2-/- hearts also had smaller LV internal end-systolic diameter (P=0.009). As LysM is also expressed in neutrophils, we also performed this study in mice where neutrophils were first depleted using an anti-Ly6G antibody. Similar cardioprotective effects were observed with MF TSC2-/- after I/R: (EF: 56.9±12.3 vs 36.1±9.8, P=0.01). This supports the impact being conferred by MF. Conclusion: TSC2-deleted MF with constitutive mTORC1 activation confer cardio-protection to I/R injury. These findings help identify a novel signaling mechanism coupling mTORC1/TSC2 in MF to myocyte recovery, highlight novel avenues to improve post ischemic cardiac repair.

Cholestasis-induced phenotypic transformation of neutrophils contributes to immune escape of colorectal cancer liver metastasis

BackgroundCholestasis is a common yet severe complication that occurs during the advancement of liver metastasis. However, how cholestasis impacts the development, treatment, and tumor microenvironment (TME) of liver metastasis remains to be elucidated.MethodsExtrahepatic and intrahepatic cholestatic mouse models with liver metastasis were established to detect the differential expression levels of genes, infiltration of immune cells and change in bile acid-associated metabolites by using RNA-Sequencing, flowcytometry, and liquid chromatography and mass spectrometry. Western blot was applied to neutrophils under the stimulation of primary bile acids (BAs) in vitro to study the mechanism of phenotypic alteration. In vitro coculture of BA-treated neutrophils with CD8+ T cells were performed to study the immune-suppressive effect of phenotypic-altered neutrophils. Clinical samples collected from colorectal cancer patients with liver metastasis and cholestasis were applied to RNA-Seq.ResultsCompared to non-cholestatic mice, the progression of liver metastasis of cholestatic mice was significantly accelerated, which was associated with increased neutrophil infiltration and T-cell exclusion. Both neutrophils and T cells expressed higher immunosuppressive markers in the cholestatic mouse model, further indicating that an immunosuppressive tumor microenvironment was induced during cholestasis. Although neutrophils deletion via anti-Ly6G antibody partially hindered liver metastasis progression, it reduced the overall survival of mice. Tauro-β-muricholic acid (Tβ-MCA) and Glycocholic acid (GCA), the two most abundant cholestasis-associated primary BAs, remarkably promoted the expression of Arg1 and iNOS on neutrophils via p38 MAPK signaling pathway. In addition, BAs-pretreated neutrophils significantly suppressed the activation and cytotoxic effects of CD8+ T cells, indicating that the immunosuppressive phenotype of neutrophils was directly induced by BAs. Importantly, targeting BA anabolism with Obeticholic acid (OCA) under cholestasis effectively suppressed liver metastasis progression, enhanced the efficacy of immune checkpoint blockade, and prolonged survival of mice.ConclusionsOur study reveals the TME of cholestasis-associated liver metastasis and proposes a new strategy for such patients by targeting bile acid anabolism.Graphical Schematic model depicting the proposed mechanism of cholestasis-mediated progression of colorectal liver metastasis.As cholestasis progresses, excessive primary bile acids that accumulate in the liver intoxicates hepatocytes, which lead to exacerbated release of chemokines, particularly CXCL2 and CXCL5. Neutrophils are then accumulated by CXCL2 and CXCL5 and undergo an immunosuppressive-phenotypic alteration induced by direct stimulation of BAs via activating the p38 MAPK signaling pathway, which eventually led to the dysfunction of T cells and progression of LM. Targeting bile acid anabolism can effectively restore the immune-activated microenvironment and prevent the progression of LM.

Abstract 3851: Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype

Abstract NAD(P)H:quinone oxidoreductase1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor selective killing. β-lapachone (β-lap), an NQO1bioactivatable drug, exhibits significant antitumor effects on NQO1+ human solid cancer cell lines. It also induces immunogenic cell death in the tumor microenvironment (TME)and synergizes with anti-PD-L1 immunotherapy. However, the interaction between β-lap-mediated anti-tumor immune response and neutrophils, a novel antigen presenting cells(APCs), remains unknown. This study found β-lap treatment increased intracellular ROS formation and DNA damage to kill NQO1+ murine tumor cell lines and dicoumarol spared the lethality. Furthermore, flow cytometric analysis of immune cells in TME of subcutaneous tumor models indicated that there is a significant infiltration of neutrophils(CD11b+ Gr1high cells) or Tumor Associated Neutrophils (TANs) following β-lap treatment. Depletion of neutrophils by co-addition of anti-Ly6G antibody abolished β-lap-induced anti-tumor efficacy in immune competent mice. Neutrophil mediated tumor cell killing determined ex vivo using bone marrow derived neutrophils and it was observed that in the presence of β-lap induced antigen, neutrophils were killing murine tumor cells. Moreover, exposure of these neutrophils to β-lap induced antigen medium induce antitumor N1-like properties, include increase in certain surface markers such as CD95(Fas) and CD54 (ICAM-1) as well as increase in intracellular cytokine IFN-β and decrease in TGF-β revealed by flow cytometric analysis. Corroborating to this, there is a significant increase of CD95 and IFN-β and a decrease in TGF-β were observed with TANs infiltrated into β-lap treated TME compared to vehicle treated tumors. Additionally, TLR/MyD88signaling deficiency decreased β-lap induced neutrophil tumor infiltration. BlockingHMGB1, depleting neutrophils, or TLR4/MyD88 deficiency lessened the antigen-specific T cell response. This study highlights that β-lap kills NQO1+ murine tumor cells through ROS production and DNA damage, and neutrophils play a pivotal role in its antitumor effects. Citation Format: Xiumei Huang, Soumya Tumbath, Liangxiang Jiang, Yang-Xin Fu. Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3851.

Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis.

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8-an anti-Ly6G antibody, or RB6-8C5-an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers-CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections.

Fusobacterium Nucleatum Aggravates Intestinal Barrier Impairment and Colitis Through IL-8 Induced Neutrophil Chemotaxis by Activating Epithelial Cells.

Inflammatory bowel disease (IBD) is affected by interactions between intestinal microbial factors, abnormal inflammation, and an impaired intestinal mucosal barrier. Neutrophils (NE) are key players in IBD. Fusobacterium nucleatum (F. nucleatum) is reported to contribute to IBD progression. However, the relationship between F. nucleatum, abnormal inflammation, and intestinal barrier impairment should be interpreted to understand the role of F. nucleatum in IBD. Dextran sulfate sodium (DSS)-induced colitis model was established and mice were orally administered with F. nucleatum. F. nucleatum colonization was confirmed by fluorescence in situ hybridization (FISH) and PCR. Intestinal barrier impairment was investigated by tight junction protein expression. Immuno-histochemistry (IHC) for Ly6G and flow cytometry detection to measure NE chemotaxis in mouse colon tissues. Caco-2 monolayers were used to evaluate epithelial integrity and permeability in vitro. A transwell model involving caco-2 cells and NE co-culture was used to assess NE chemotaxis. NE chemokines were measured by ELISA. A mouse model of NE exhaustion using an anti-Ly6G antibody was used to identify the role of NEs in F. nucleatum-induced colitis. Transcriptome sequencing and bioinformatics analysis were applied to screen cytokines and signaling pathways. Administration of F. nucleatum aggravated colitis in the DSS model. F. nucleatum infection downregulates ZO-1 and Occludin expression and increases intestinal permeability. Additionally, F. nucleatum-induced NE chemotaxis decreases the integrity and permeability of the caco-2 monolayer. F. nucleatum-induced NE chemotaxis is dependent on IEC-derived interleukin 8 (IL-8) secretion, mediated by the TLR2/ERK signaling pathway. In addition, NE exhaustion in mice inhibited F. nucleatum-induced intestinal barrier impairment and colitis. F. nucleatum improves NE chemotaxis by infecting intestinal epithelial cells (IECs) to secrete IL-8 and aggravate intestinal barrier impairment, contributing to the progression of intestinal inflammation. Examining and eliminating F. nucleatum could be a valuable microbiome-based method for IBD surveillance and prevention.

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamycin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.

AT 1 inhibition mediated neuroprotection after experimental traumatic brain injury is dependent on neutrophils in male mice

After traumatic brain injury (TBI) cerebral inflammation with invasion of neutrophils and lymphocytes is a crucial factor in the process of secondary brain damage. In TBI the intrinsic renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) reduces secondary brain damage and the invasion of neutrophil granulocytes into injured cerebral tissue. The current study explored the involvement of immune cells in neuroprotection mediated by AT1 inhibition following experimental TBI. Four different cohorts of male mice were examined, investigating the effects of neutropenia (anti-Ly6G antibody mediated neutrophil depletion; C57BL/6), lymphopenia (RAG1 deficiency, RAG1−/−), and their combination with candesartan-mediated AT1 inhibition. The present results showed that reduction of neutrophils and lymphocytes, as well as AT1 inhibition in wild type and RAG1−/− mice, reduced brain damage and neuroinflammation after TBI. However, in neutropenic mice, candesartan did not have an effect. Interestingly, AT1 inhibition was found to be neuroprotective in RAG1−/− mice but not in neutropenic mice. The findings suggest that AT1 inhibition may exert neuroprotection by reducing the inflammation caused by neutrophils, ultimately leading to a decrease in their invasion into cerebral tissue.

Leukemic Environment Alters Neutrophils Worsening Disease Progression

Abstract Acute Myeloid Leukemia (AML) is the abnormal differentiation of hematopoietic stem cells in the bone marrow into malignant blasts. The leukemic marrow environment plays an important role during disease progression, by shaping the immune landscape interacting with these blasts. Mounting evidence suggests neutrophils are complicit within tumors by enhancing tumorigenesis and immunosuppression; however, little is known about how these cells contribute to AML even though neutrophils comprise half the marrow cellularity. Tumor-associated neutrophils are found in an overactivated state, constantly releasing pro-inflammatory cytokines and granular proteins to promote tumorigenesis. Therefore, we investigated the neutrophil phenotype in our AML chimeric mouse model. We observed neutrophils in AML were also in an activated state. More importantly, sorted leukemic-exposed neutrophils were more susceptible to degranulation upon stimulation. Next, our single-cell RNA data showed that neutrophils within the AML environment had higher expression of ELANE, a gene encoding neutrophil elastase (NE). Increased NE levels have been associated with increased tumor burden in many cancers. Although NE levels increase inflammation which drives tumorigenesis, it may also directly increase leukemic blast proliferation. By targeting neutrophils in our AML chimeric model using anti-Ly6G antibody, we found decreased frequency of leukemic blasts in the neutrophil depleted mice. Also, there was a reduced frequency of the Lin-ckit+ CD16/32+ leukemic stem cells. We hypothesize that over-activated neutrophils drive AML progression in the BM by releasing NE, providing a new avenue for disease intervention.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

Abstract 1353: CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow

Abstract Tumors emerge and evolve in complex and dynamic microenvironments that influence their growth, invasion, and metastasis. In the tumor microenvironment, the immune system plays a critical role in the prevention and elimination of tumors. Despite immune surveillance, tumors still manage to develop through evolution facilitating the establishment of an immune suppressive microenvironment. There is emerging evidence showing that the antitumor immunity of immune effector cells can be obstructed by immune suppressive cell populations such as MDSCs or Tregs. Hematologic malignancies associated with FGFR1 translocations, also known as Stem Cell Leukemia/Lymphoma (SCLL) syndrome, show a complex presentation including myeloproliferative neoplasm, AML, and T- or B-cell lineage lymphoblastic leukemia/lymphoma, providing an ideal, homogeneous model to investigate mechanisms underlying leukemogenesis. Our recent investigation in mouse models of SCLL revealed that mice xenografted with SCLL cells showed increased CD11b+Ly6CHigh M-MDSC and CD11b+Ly6CInt PMN-MDSC levels in the peripheral blood (PB) during leukemia development, accompanied by significantly decreased levels of T cells. To further confirm this MDSC-mediated suppression of T cell function, MDSC depletion experiments were performed in the SCLL cell engraftment model using either the anti-Ly6G antibody (for PMN-MDSCs) or the anti-Gr-1 (aka Ly6G/Ly6C; for total MDSCs) antibody. In both cases, significantly increased levels of CD4+ and CD8+ cells were observed, along with significantly prolonged mouse survival. These observations were consistent in multiple SCLL cell line models, supporting the idea that SCLL cells promote the accumulation of MDSCs, which in turn suppresses the antitumor activity of cytotoxic T cells, allowing SCLL progression. To further characterize these leukemia cell induced MDSCs, CyTOF analysis was performed with the PBMC and bone marrow (BM) samples from leukemic and naïve mice. In peripheral blood leukemia cell induced M-MDSCs also express macrophage markers, including F4-80 and CD64, implying a potential overlap of MDSC and tumor-associated macrophage (TAM). These TAMs also express high CD71 and CD184. Most importantly, we can also identify a CD64/CD71/CD184 expressing cluster in the BM samples. Different from the PB, these cells in the BM express stem cell marker CD117, suggesting there are potential progenitor cells of TAMs. Taken together, our studies demonstrated that SCLL cells establish a tumor favorable microenvironment by reshaping hematopoiesis toward MDSC generation in the BM. A comprehensive understanding of these immune modulation processes holds the promise of improving patient outcomes by reversing the generation of MDSC/TAM in the BM and overturning the establishment of the immunosuppressive microenvironment at the outset. Citation Format: Xiaocui Lu, Ting Zhang, Atsuko Matsunaga, Xuexiu Fang, Stephanie F. Mori, John Cowell, Tianxiang Hu. CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1353.

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 µg of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.