Influx Of Monocytes Research Articles

Disruption of the C5a/C5aR1 axis confers protection against hookworm infection in lung.

Hookworms are soil-transmitted parasitic nematodes that penetrate the host skin before migrating to the lungs. With an estimated 500-700 million people infected worldwide, hookworm infections are a neglected tropical disease and a significant cause of morbidity, particularly in children, pregnant women, and immunocompromised individuals. Although there is ample evidence that complement activation is pivotal to elicit a protective host immune response against invasive pathogens, its role in hookworm infection remains insufficiently explored. Here, we investigated the complement anaphylatoxin, C5a, during the early lung stage of infection with Nippostrongylus brasiliensis in C57BL/6J wild type and C5aR1-/- mice. Despite the previously reported ability of lung larvae to evade complement activation, C5a was detectable locally in lung tissue and bronchoalveolar lavages. Surprisingly, C5aR1 presence directly contributed to the pathogenicity of hookworm infection. The burden of viable parasites in the lungs was mitigated in C5aR1-/- mice, compared to C57BL/6J mice 48 hours post-infection. Additionally, C5aR1-/- mice showed significantly reduced lung injury, lower cytokine release, attenuated alveolar hemorrhage, and limited alveolar-capillary barrier disruption. Neutrophils were the most abundant and highest C5aR1-expressing cell type in the alveolar space after infection. Deficiency of C5aR1 reduced the influx of neutrophils, monocytes, and eosinophils to the pulmonary airways. RNA sequencing of alveolar neutrophils revealed C5aR1-dependent regulation of the novel nuclear protein, DEDD2. In conclusion, our findings highlight the impact of C5aR1 signaling in neutrophils during hookworm infection uncovering an unexpected downside of complement activation in parasitic infection.

Cardiac Repair after Myocardial Infarction is Controlled by a Complement C5a Receptor 1-Driven Signaling Cascade.

Cardiac repair following myocardial infarction is important in regenerating functionally viable myocardium to prevent cardiac death. Previous studies have linked C5aR1 to cardiac regeneration and inflammation. However, C5a receptor-driven responses during the late phases, up to 4 weeks of the infarction insult - a time window that specifically reflects the outcome of the repair process - and the underlying mechanisms are poorly defined. Here, we show that C5ar1, but not C5ar2, deficiency attenuates infarct size following coronary artery ligation-induced myocardial infarction (MI). C5ar1 deficiency limited the deposition of collagen and mitigated cell death in infarcted areas leading to overall improved cardiac function four weeks after MI. While infiltration of neutrophils was reduced in both C5ar1-/- and C5ar2-/- infarcted myocardium 24 h after MI, influx of monocytes after 1 week of MI was reduced only upon C5ar1 deficiency. Subsequent analysis revealed reduced accumulation of myofibroblast, elevated expression of transforming growth factor-beta1 (Tgf-β1) and vascular endothelial growth factor-A (Vegf-A) in C5ar1-/- infarcted hearts. In vitro, exogenous TGF-β1 triggered conversion of fibroblasts into myofibroblasts, which expressed Vegf-A mRNA - an effect that was enhanced upon C5ar1 deficiency. Incubation of C5ar1+/+ or C5ar1-/- endothelial cells (ECs) with supernatants from C5ar1+/+ or C5ar1-/- myofibroblasts respectively, mimicking the in vivo microenvironment in our model, led to significant increase in matrigel tube formation in C5ar1-/- ECs. This was consistent with enhanced neoangiogenesis in C5ar1-/- infarcted hearts. Collectively, our study demonstrates that inhibition of C5aR1 has the potential to improve cardiac function after myocardial infarction.

Acetylsalicylic acid and dihydroartemisinin combined therapy on experimental malaria-associated acute lung injury: analysis of lung function and the inflammatory process

BackgroundSevere malaria can cause respiratory symptoms, which may lead to malaria-acute lung injury (MA-ALI) due to inflammation and damage to the blood-gas barrier. Patients with severe malaria also often present thrombocytopenia, and the use of acetylsalicylic acid (ASA), a commonly used non-steroidal anti-inflammatory drug with immunomodulatory and antiplatelet effects, may pose a risk in regions where malaria is endemic. Thus, this study aimed to investigate the systemic impact of ASA and dihydroartemisinin (DHA) on ALI induced in mice by Plasmodium berghei NK65 (PbNK65).MethodsC57BL/6 mice were randomly divided into control (C) and PbNK65 infected groups and were inoculated with uninfected or 104 infected erythrocytes, respectively. Then, the animals were treated with DHA (3 mg/kg) or vehicle (DMSO) at the 8-day post-infection (dpi) for 7 days and with ASA (100 mg/kg, single dose), and analyses were performed at 9 or 15 dpi. Lung mechanics were performed, and lungs were collected for oedema evaluation and histological analyses.ResultsPbNK65 infection led to lung oedema, as well as increased lung static elastance (Est, L), resistive (ΔP1, L) and viscoelastic (ΔP2, L) pressures, percentage of mononuclear cells, inflammatory infiltrate, hemorrhage, alveolar oedema, and alveolar thickening septum at 9 dpi. Mice that received DHA or DHA + ASA had an increase in Est, L, and CD36 expression on inflammatory monocytes and higher protein content on bronchoalveolar fluid (BALF). However, only the DHA-treated group presented a percentage of inflammatory monocytes similar to the control group and a decrease in ΔP1, L and ΔP2, L compared to Pb + DMSO. Also, combined treatment with DHA + ASA led to an impairment in diffuse alveolar damage score and lung function at 9 dpi.ConclusionsTherapy with ASA maintained lung morpho-functional impairment triggered by PbNK65 infection, leading to a large influx of inflammatory monocytes to the lung tissue. Based on its deleterious effects in experimental MA-ALI, ASA administration or its treatment maintenance might be carefully reconsidered and further investigated in human malaria cases.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis. Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.

Kinetics of infection and local immune response in a model of chlamydia trachomatis conjunctival infection

Aims/Purpose: Trachoma is caused by Chlamydia trachomatis conjunctival infections and evolves in two phases: i) active or acute trachoma caused by repeated infections and ii) scarring or chronic trachoma leading to conjunctival scarring, eyelid malposition, and corneal opacification. It remains the first cause of infectious blindness in developing countries. The purpose of this study was to precisely describe the kinetics of infection and local immune response in a non‐human primate model of acute trachoma.Methods: Cynomolgus Macaques received a single conjunctival inoculation of B/Tunis‐864 strain (104 IFU) and were assessed at weeks 2, 4, 6, 9, and 11. Clinical evaluation was performed by 3 independent masked observers on standardized ocular surface photographs graded using specific scores for both inflammation and follicles. Local immune response was assessed using multiparameter flow cytometry of conjunctival cells eluted from conjunctival prints. Tears cytokines were quantified using bead‐based Luminex multiplex assay. Bacterial quantification in conjunctival fluids was performed with quantitative PCR.Results: All animals (n = 12) developed typical follicular conjunctivitis (n = 12). The first 4 weeks post‐exposure were characterized by a conjunctival T cell influx (63% of CD45+ cells at week 3), with elevated interleukins 4, 8, and 5. In the later phase, after 9 weeks, we observed an influx of monocytes (53,2% of CD45+ cells at week 11) with reduced immune cells and a return to the initial cytokines level.Conclusion: This model faithfully mimics trachoma's acute phase. Local immunologic assays showed an early lymphocyte response with long‐term persistence of innate immune effectors. It offers an opportunity to assess potential preventive therapeutic strategies. Further studies, including implementing repeated bacterial conjunctival inoculations, are warranted to investigate the later stages of the diseases.

Prevention of severe lung immunopathology associated with influenza infection through adeno-associated virus vector administration

BackgroundInfluenza A viruses (IAVs) have long posed a threat to humans, occasionally causing significant morbidity and mortality. The initial immune response is triggered by infected epithelial cells, alveolar macrophages and dendritic cells. However, an exaggerated innate immune response can result in severe lung injury and even host mortality. One notable pathology observed in hosts succumbing to severe influenza is the excessive influx of neutrophils and monocytes into the lung. In this study, we investigated a strategy for controlling lung immunopathology following severe influenza infection.ResultsTo evaluate the impact of innate immunity on influenza-associated lung injury, we employed CB17.SCID and NOD.SCID mice. NOD.SCID mice exhibited slower weight loss and longer survival than CB17.SCID mice following influenza infection. Lung inflammation was reduced in NOD.SCID mice compared to CB17.SCID mice. Bulk RNA sequencing analysis of lung tissue showed significant downregulation of 827 genes, and differentially expressed gene analysis indicated that the cytokine-cytokine receptor interaction pathway was predominantly downregulated in NOD.SCID mice. Interestingly, the expression of the Cxcl14 gene was higher in the lungs of influenza-infected NOD.SCID mice than in CB17.SCID mice. Therefore, we induced overexpression of the Cxcl14 gene in the lung using the adeno-associated virus 9 (AAV9)-vector system for target gene delivery. However, when we administered the AAV9 vector carrying the Cxcl14 gene or a control AAV9 vector to BALB/c mice from both groups, the morbidity and mortality rates remained similar. Both groups exhibited lower morbidity and mortality than the naive group that did not receive the AAV9 vector prior to IAV infection, suggesting that the pre-administration of the AAV9 vector conferred protection against lethal influenza infection, irrespective of Cxcl14 overexpression. Furthermore, we found that pre-inoculation of BALB/c mice with AAV9 attenuated the infiltration of trans-macrophages, neutrophils and monocytes in the lungs following IAV infection. Although there was no difference in lung viral titers between the naive group and the AAV9 pre-inoculated group, pre-inoculation with AAV9 conferred lung injury protection against lethal influenza infection in mice.ConclusionsOur study demonstrated that pre-inoculation with AAV9 prior to IAV infection protected mouse lungs from immunopathology by reducing the recruitment of inflammatory cells.

FTY720 attenuates acute colitis via colonic T cells reduction and inhibition of M1 macrophages polarization independent of CCR2-mediated monocytes input

Ulcerative colitis (UC) is a complex multifactorial disease, of which the exact etiology is not fully understood. The inappropriate aggressive inflammatory response is closely related to the disease progression of UC. FTY720 is a sphingosine-1-phosphate receptor agonist and acts as a key immunomodulator in inflammation. This study aims to investigate the protective influence of FTY720 on inflammation in the DSS-induced colitis model. In the present study, the C57BL/6 mice and the CCR2−/− mice were exposed to 5% Dextran Sodium Sulfate (DSS) drinking water for 6 days followed by an injection of FTY720 (1 mg/kg/d) or vehicle (PBS) 6 times starting on the next day. The body weight, stool consistency, and occult blood were assessed daily. The inflammatory cytokines level in colon tissues and serum were assessed. Leukocyte subsets of bone marrow (BM), spleen, and colon were analyzed by flow cytometry. Our results demonstrated that FTY720 ameliorated the aberrant immune responses by trapping T cells and inhibiting the polarization of M1 macrophages in colitis mice. The effect of FTY720 on the increased number of colonic macrophages did not dependent on CCR2-mediated monocyte influx, despite most monocytes being reduced after DSS administration in the inflamed colon of CCR2−/− mice. Rather, depletion of CCR2 did not impact the protective influence of FTY720 on colonic injury in acute colitis. All these findings unravel a beneficial function of FTY720 in the inflammatory response to DSS-induced acute colitis, provided further insights into monocyte migration and might provide potential opportunities for UC therapeutic intervention.

Pathomechanism of the IVDs Degeneration and the Role of Neurotrophic Factors and Concentration of Selected Elements in Genesis of Low Back Pain.

Degenerative disc disease of the lumbosacral spine is a very common medical problem. An episode of sciatica occurs at least once in the life of 60-90% of the human population. A phenomenon that is closely related to the process of lowering the pH of the extracellular matrix degenerating the intervertebral disc (IVD) is the precipitation of calcium salts, especially pyrophosphate dehydrate and hydroxyapatite. In such an altered environment of the IVD, we can observe an increased influx of monocytes, macrophages, T-lymphocytes, as well as non-immunocompetent cells, which are a source of cytokines, e.g., tumor necrosis alpha (TNF-α), interleukin- (IL-1β, IL-8). The above-mentioned mediators of an inflammatory condition contribute to an increase in the expression of Brain-Derived Neurotrophic Factor (BDNF) and Glial cell Derived Neurotrophic Factor (GDNF) in mast cells and chondrocytes, as well as to the descending transport of these mediators along the nerve endings. In the process of degeneration of the IVD as a result of repeated and even slight injuries, there is damage to the connections of the endplate of the vertebral bodies with the IVD, which results in an impairment of the penetration of nutritional substances and water into the disc. As a consequence, there is an overexpression of the brain-derived neurotrophic factor GDNF, as well as neuromodulin (GAP-43) in the mast cells and chondrocytes of the IVDs, while descending transport of these mediators along the nerve fibers is also observed.

Tubulointerstitial nephritis antigen-like 1 is a novel matricellular protein that promotes gastric bacterial colonization and gastritis in the setting of Helicobacter pylori infection.

The interaction between the gastric epithelium and immune cells plays key roles in H. pylori-associated pathology. Here, we demonstrate a procolonization and proinflammatory role of tubulointerstitial nephritis antigen-like 1 (TINAGL1), a newly discovered matricellular protein, in H. pylori infection. Increased TINAGL1 production by gastric epithelial cells (GECs) in the infected gastric mucosa was synergistically induced by H. pylori and IL-1β via the ERK-SP1 pathway in a cagA-dependent manner. Elevated human gastric TINAGL1 correlated with H. pylori colonization and the severity of gastritis, and mouse TINAGL1 derived from non-bone marrow-derived cells promoted bacterial colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Tinagl1-/- and Tinagl1ΔGEC mice and were increased in mice injected with mouse TINAGL1. Mechanistically, TINAGL1 suppressed CCL21 expression and promoted CCL2 production in GECs by directly binding to integrin α5β1 to inhibit ERK and activate the NF-κB pathway, respectively, which not only led to decreased gastric influx of moDCs via CCL21-CCR7-dependent migration and, as a direct consequence, reduced the bacterial clearance capacity of the H. pylori-specific Th1 response, thereby promoting H. pylori colonization, but also resulted in increased gastric influx of Ly6Chigh monocytes via CCL2-CCR2-dependent migration. In turn, TINAGL1 induced the production of the proinflammatory protein S100A11 by Ly6Chigh monocytes, promoting H. pylori-associated gastritis. In summary, we identified a model in which TINAGL1 collectively ensures H. pylori persistence and promotes gastritis.

The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis.

The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli. A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function.

Morphine-mediated release of astrocyte-derived extracellular vesicle miR-23a induces loss of pericyte coverage at the blood-brain barrier: Implications for neuroinflammation.

Opioids such as morphine are the most potent and efficacious drugs currently available for pain management. Paradoxically, opioids have also been implicated in inducing neuroinflammation and associated neurocognitive decline. Pericytes, a critical component of the neurovascular unit (NVU), are centrally positioned between endothelial cells and astrocytes, maintaining function of the blood-brain barrier (BBB) nd regulating neuroinflammation by controlling monocyte influx under various pathological conditions. The role of pericytes in morphine-mediated neuroinflammation however, has received less attention, especially in the context of how pericytes crosstalk with other central nervous system (CNS) cells. The current study was undertaken to examine the effect of miRNAs released from morphine-stimulated human primary astrocyte-derived extracellular vesicles (morphine-ADEVs) in mediating pericyte loss at the blood-brain barrier, leading, in turn, to increased influx of peripheral monocytes. Our findings suggest that the heterogeneous nuclear ribonucleoprotein complex A2/B1 (hnRNP A2/B1) plays role in morphine-mediated upregulation and release of miR-23a in ADEVs, and through action of morphine via mu opioid receptor.We further demonstrated that miR-23a in morphine-ADEVs could be taken up by pericytes, resulting in downregulation of PTEN expression, ultimately leading to increased pericyte migration. Furthermore, both overexpression of PTEN and blocking the miR-23a target site at PTEN 3UTR (by transfecting miR-23a-PTEN target protector), attenuated morphine-ADEV-mediated pericyte migration. We also demonstrated that in the microvessels isolated from morphine-administered mice, there were fewer PDGFβR + pericytes co-localizing with CD31+ brain endothelial cells compared with those from saline mice. In line with these findings, we also observed increased loss of pericytes and a concomitantly increased influx of monocytes in the brains of morphine-administered pericyte-labeled NG2-DsRed mice compared with saline mice. In conclusion, our findings indicate morphine-ADEVs mediated loss of pericyte coverage at the brain endothelium, thereby increasing the influx of peripheral monocytes in the central nervous system, leading to neuroinflammation.

Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model.

Background: Gout is a common arthritis, due to deposition of monosodium urate (MSU) crystals which results in IL-1β secretion by tissue-resident macrophages. Xanthine oxidase (XO) catalyzes uric acid (UA) production and in the process, reactive oxygen species (ROS) are generated which contributes to NLRP3 inflammasome activation. Protein phosphatase 2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. The objective of this study was to investigate whether PP2A regulates gout inflammation, mediated by XO activity modulation. We studied UA and ROS generations in MSU stimulated murine bone marrow derived macrophages (BMDMs) in response to fingolimod phosphate, a PP2A activator, and compared its anti-inflammatory efficacy to that of an XO inhibitor, febuxostat. Methods: BMDMs were stimulated with MSU, GM-CSF/IL-1β or nigericin ± fingolimod (2.5μM) or febuxostat (200μM) and UA levels, ROS, XO, and PP2A activities, Xdh (XO) expression and secreted IL-1β levels were determined. PP2A activity and IL-1β in MSU stimulated BMDMs ± N-acetylcysteine (NAC) (10μM) ± okadaic acid (a PP2A inhibitor) were also determined. M1 polarization of BMDMs in response to MSU ± fingolimod treatment was assessed by a combination of iNOS expression and multiplex cytokine assay. The in vivo efficacy of fingolimod was assessed in a murine peritoneal model of acute gout where peritoneal lavages were studied for pro-inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils by flow cytometry and IL-1β by ELISA. Results: Fingolimod reduced intracellular and secreted UA levels (p < 0.05), Xdh expression (p < 0.001), XO activity (p < 0.001), ROS generation (p < 0.0001) and IL-1β secretion (p < 0.0001), whereas febuxostat enhanced PP2A activity (p < 0.05). NAC treatment enhanced PP2A activity and reduced XO activity and PP2A restoration mediated NAC's efficacy as co-treatment with okadaic acid increased IL-1β secretion (p < 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p < 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p < 0.001). Fingolimod reduced iNOS expression (p < 0.0001) and secretion of IL-6 and TNF-α (p < 0.05). Fingolimod reduced CMs (p < 0.0001), neutrophil (p < 0.001) and IL-1β (p < 0.05) lavage levels while increasing NCMs (p < 0.001). Conclusion: Macrophage PP2A is inactivated in acute gout by ROS and a PP2A activator exhibited a broad anti-inflammatory effect in acute gout in vitro and in vivo.

Buprenorphine reverses neurocognitive impairment in EcoHIV infected mice: A potential therapy for HIV-NCI.

Thirty-eight million people worldwide are living with HIV, PWH, a major public health problem. Antiretroviral therapy (ART) revolutionized HIV treatment and significantly increased the lifespan of PWH. However, approximately 15-50% of PWH develop HIV associated neurocognitive disorders (HIV-NCI), a spectrum of cognitive deficits, that negatively impact quality of life. Many PWH also have opioid use disorder (OUD), and studies in animal models of HIV infection as well as in PWH suggest that OUD can contribute to HIV-NCI. The synthetic opioid agonist, buprenorphine, treats OUD but its effects on HIV-NCI are unclear. We reported that human mature inflammatory monocytes express the opioid receptors MOR and KOR, and that buprenorphine reduces important steps in monocyte transmigration. Monocytes also serve as HIV reservoirs despite effective ART, enter the brain, and contribute to HIV brain disease. Using EcoHIV infected mice, an established model of HIV infection and HIV-NCI, we previously showed that pretreatment of mice prior to EcoHIV infection reduces mouse monocyte entry into the brain and prevents NCI. Here we show that buprenorphine treatment of EcoHIV infected mice with already established chronic NCI completely reverses the disease. Disease reversal was associated with a significant reduction in brain inflammatory monocytes and reversal of dendritic injury in the cortex and hippocampus. These results suggest that HIV-NCI persistence may require a continuing influx of inflammatory monocytes into the brain. Thus, we recommend buprenorphine as a potential therapy for mitigation of HIV brain disease in PWH with or without OUD.

Monocyte-derived peritoneal macrophages protect C57BL/6 mice against surgery-induced adhesions.

Peritoneal adhesions commonly occur after abdominal or pelvic surgery. These scars join internal organs to each other or to the cavity wall and can present with abdominal or pelvic pain, and bowel obstruction or female infertility. The mechanisms underlying adhesion formation remain unclear and thus, effective treatments are not forthcoming. Peritoneal macrophages accumulate after surgery and previous studies have attributed either pro- or anti-scarring properties to these cells. We propose that there are complex and nuanced responses after surgery with respect to both resident and also monocyte-derived peritoneal macrophage subpopulations. Moreover, we contend that differences in responses of specific macrophage subpopulations in part explain the risk of developing peritoneal scars. We characterized alterations in peritoneal macrophage subpopulations after surgery-induced injury using two strains of mice, BALB/c and C57BL/6, with known differences in macrophage response post-infection. At 14 days post-surgery, BALB/c mice displayed more adhesions compared with C57BL/6 mice. This increase in scarring correlated with a lower influx of monocyte-derived macrophages at day 3 post-surgery. Moreover, BALB/c mice showed distinct macrophage repopulation dynamics after surgery. To confirm a role for monocyte-derived macrophages, we used Ccr2-deficient mice as well as antibody-mediated depletion of CCR2 expressing cells during initial stages of adhesion formation. Both Ccr2-deficient and CCR2-depleted mice showed a significant increase in adhesion formation associated with the loss of peritoneal monocyte influx. These findings revealed an important protective role for monocyte-derived cells in reducing adhesion formation after surgery.

A shift in lung macrophage composition is associated with COVID-19 severity and recovery.

Although it has been more than 2 years since the start of the coronavirus disease 2019 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, therapies to treat COVID-19 and other inflammatory diseases remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and mortality to develop tailored immunotherapy strategies to halt disease progression. We assembled the Mount Sinai COVID-19 Biobank, which was composed of almost 600 hospitalized patients followed longitudinally through the peak of the pandemic in 2020. Moderate disease and survival were associated with a stronger antigen presentation and effector T cell signature. In contrast, severe disease and death were associated with an altered antigen presentation signature, increased numbers of inflammatory immature myeloid cells, and extrafollicular activated B cells that have been previously associated with autoantibody formation. In severely ill patients with COVID-19, lung tissue-resident alveolar macrophages not only were drastically depleted but also had an altered antigen presentation signature, which coincided with an influx of inflammatory monocytes and monocyte-derived macrophages. In addition, we found that the size of the alveolar macrophage pool correlated with patient outcome and that alveolar macrophage numbers and functionality were restored to homeostasis in patients who recovered from COVID-19. These data suggest that local and systemic myeloid cell dysregulation are drivers of COVID-19 severity and modulation of alveolar macrophage numbers and activity in the lung may be a viable therapeutic strategy for the treatment of critical inflammatory lung diseases.

Stem cell factor/mast cell/CCL2/monocyte/macrophage axis promotes Coxsackievirus B3 myocarditis and cardiac fibrosis by increasing Ly6Chigh monocyte influx and fibrogenic mediators production.

Mast cells (MCs), central players in allergy and parasitic infections, play key roles in inflammation and fibrosis. Here, the impact of MCs on the progression of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) and fibrosis was investigated using MC-deficient KitW-sh mice. Viral titres, cellular infiltrates and heart pathologies were evaluated and compared with wild-type (WT) mice during acute CVB3 infection of C57BL/6 mice. CVB3 infection induced an increased accumulation and degranulation of MCs in the hearts of mice during acute infection. MC-deficient KitW-sh mice had slightly higher viral titres, decreased VMC and cardiac fibrosis and improved cardiac dysfunction compared to WT mice via decreasing cardiac influx of Ly6Chigh monocytes/macrophages (Mo/Mφ). While bone marrow-derived MC reconstitution decreased viral titre and worsened improved survival and VMC severity in Wsh mice. MC-fibroblasts co-culture revealed a cardiac MC-fibroblasts crosstalk during early infection: fibroblasts trigger MC degranulation and secretion of CCL2 and tumour necrosis factor alpha (TNF-α) via producing early stem cell factor (SCF); while MCs-fibrogenic mediators (TNF-α) stimulate fibroblasts to increase CCL2, α-smooth muscle actin (SMA), collagen and transforming growth factor beta（TGFβ)expression, thus aggravating cardiac fibrosis. MCs and fibroblast-derived CCL2s are both essential for cardiac Ly6Chigh Mo/Mφ influx. Administration of recombinant mouse SCF to CVB3-infected mice aggravates VMC via accelerating MCs accumulation and cardiac influx of Ly6Chi Mo/Mφ. Collectively, our data highlight an early MC-fibroblast crosstalk and SCF/MC/CCL2/Mo/Mφ axis as important mechanisms required for triggering VMC and myocardial fibrosis. This finding indicates critical roles of MCs in initiating and modulating cardiac innate response to CVB3 and has an implication in developing new and more effective treatments for VMC.

Widespread retina and optic nerve neuroinflammation in enucleated eyes from glaucoma patients

Neuroinflammation is recognized as a key component of neurodegenerative disease. In glaucoma, a common neurodegenerative disease and the leading cause of irreversible blindness, the evidence for neuroinflammation in patients is lacking. Animal models have demonstrated significant pro-inflammatory activation of resident glia in the retina, as well as influx of blood-derived monocytes and pro-inflammatory factors. Confirmation of this in human donor tissue has been challenging due to a lack of well-preserved and well-characterized post-mortem tissue. To address this we utilize archived, wax embedded eyes fixed immediately following enucleation from living glaucoma patients. We compared glaucoma to control eyes (enucleated for uveal melanoma where the tumor did not impact the central retina or optic nerve). We performed immunolabelling for neurodegenerative and glial markers (CD45, CD163, IBA1, GFAP, Vimentin) which were quantified by high-resolution light microscopy and image analysis in FIJI. Glaucoma eyes demonstrated significant neural loss consistent with advanced neurodegeneration. IBA1 and GFAP were significantly increased in the retina and optic nerve head of the glaucomatous eyes indicating that significant neuroinflammation had occurred which support findings in animal models. Inflammation is a treatable symptom of many diseases and as such, identification of earlier inflammatory processes in glaucoma could be important for potential future treatment options.

Turnover Kinetics of Pancreatic Macrophages in Lean and Obese Diabetic Mice.

Pancreatic resident macrophages, a heterogeneous family of cells with distinct origins and phenotypes, are the main myeloid cells in exocrine and endocrine tissues. Adult exocrine F4/80hi macrophages consist of three different subsets based on the embryonic marker Tim-4 and MHC II expression. Their frequencies shift during aging and obesity with the Tim-4-MHCII+ fraction becoming the predominant subpopulation in the inter acinar stroma. Endocrine resident F4/80hi macrophages are more homogenous and represent the prevalent leukocyte fraction residing within the islets in both lean and obese mice. We used an adult fate mapping mouse model to characterize turnover kinetics within the pancreatic resident macrophages under normal homeostasis and obese diabetic conditions. We demonstrate that islet resident macrophages show unique replenishment kinetics, with embryonic macrophages being gradually replaced by bone marrow-derived monocytes with increasing age. Their replenishment was independent of the CCL2/CCR2 axis. Furthermore, we confirmed that both exocrine Tim-4+MHCIIlow and Tim-4+MHCII+ fractions are long-lived and primarily independent from bone marrow-derived monocytes. In contrast, exocrine Tim-4-MHCII+ macrophages are gradually replaced through a CCR2-dependent influx of bone marrow-derived monocytes in aging. Moreover, we show that obesity and type 2 diabetes do not affect the turnover kinetics of any macrophage subpopulation residing in the pancreas. Our study uncovers new insights on pancreatic macrophage biology in aging and obesity.

EPEN-26. Chemokine receptor blockade reverses CCL2 mediated immunosuppression and restores CAR-T cell function in posterior fossa ependymoma

Trastuzumab-based HER2 CAR-T constructs have demonstrated preclinical efficacy in medulloblastoma and are being evaluated for use in children and young adults with recurrent or refractory CNS tumors. Preliminary results demonstrate immune activation but no objective tumor response in three patients, including two patients with posterior fossa (PF)-EPN. A key finding in the serum and CSF of all three patients was very high levels of the inflammatory chemokine CCL2 following treatment with CAR-T cells. Preclinical studies suggest that high levels of CCL2 may impede T cell mediated anti-tumor activity in CNS tumors. The role of CCL2 to enhance or diminish CAR-T cell efficacy for CNS tumors is unknown. We evaluated a second generation trastuzumab-based HER2 CAR construct with a 4-1BB co-stimulatory domain in two ultra-high-risk patient-derived xenograft (PDX) models that faithfully recapitulate PFA-EPN. In contrast to preclinical studies in other cancers, treatment with trastuzumab-based HER2 CAR-T cell alone causes only partial regression of tumors and robust infiltration of immunosuppressive monocytes in PFA-EPN PDX mouse models. We studied constitutive NF-kB activation because it is a hallmark of PFA-EPN that drives dysregulation of inflammatory genes and forms an immunosuppressive tumor microenvironment. Upon tumor recognition, CAR-T cells produce high amounts of the cytokine tumor necrosis factor-alpha, which is an extracellular stimulus that propagates NF-kB activation in PFA-EPN. We show that HER2 CAR-T cell treatment causes increased nuclear translocation of the RELA NF-kB subunit, which induces CCL2 gene transcription and chemokine release. This results in CCL2-CCR2 ligand/receptor mediated influx of inflammatory monocytes and regulatory T cells, impairing CAR-T cell effector function. Inhibition of CCR2 restores anti-tumor CAR-T cytotoxicity against bulky orthotopic tumors by decreasing the infiltration of inflammatory monocytes and regulatory T cells. Combinatorial strategies addressing tumor mediated immunosuppression should be evaluated in upcoming CAR-T cell trials for patients with high-risk CNS tumors.