Two subsets of circulating Ly6Clo monocytes distinguished by CD138 (syndecan-1) expression and Nr4a1 dependence in pristane-treated mice

Chronic peritoneal inflammation following pristane injection induces lupus with diffuse alveolar hemorrhage (DAH) and pulmonary capillaritis in C57BL/6 mice. The pathogenesis involves pristane-induced microvascular lung injury. BALB/c mice are resistant to endothelial injury and DAH. Lung disease in C57BL/6 mice is abolished by depleting monocytes/macrophages. The objective of this study was to define the role of myeloid subsets in DAH. Hemorrhage and vasculitis were abolished in Ccr2-/- mice, indicating involvement of bone marrow-derived monocytes/macrophages. Along with Ly6Chi monocytes, we found two subsets of circulating Ly6Clo monocytes: one CD138- and a novel CD138+ subset. Nr4a1-dependent patrolling Ly6Clo monocytes maintain vascular integrity after endothelial injury. Circulating Ly6CloCD138+ monocytes were associated with DAH and were absent in mice without DAH. They also were absent in Nr4a1-/- mice, whereas Ly6CloCD138- monocytes were unaffected. However, Nr4a1-/- mice were susceptible to pristane-induced DAH and lung vasculitis, suggesting that disease onset does not require Ly6CloCD138- monocytes. Peritoneal Ly6CloCD138+ M? were unchanged in Nr4a1-/- mice, indicating that they are not derived from Ly6CloCD138+ monocytes. We conclude that pristane-induced lung microvascular lung injury stimulates a wave of Nr4h1-dependent Ly6CloCD138+ patrolling monocytes in an ineffectual effort to maintain vascular integrity in the face of ongoing endothelial damage.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Anti-TLR7 Antibody Protects Against Lupus Nephritis in NZBWF1 Mice by Targeting B Cells and Patrolling Monocytes

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6Clow patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.

Intravascular Crawling of Patrolling Monocytes: A Lèvy-Like Motility for Unique Search Functions?

Patrolling monocytes (PMo) are the organism’s preeminent intravascular guardians by their continuous search of damaged endothelial cells and harmful microparticles for their removal and to restore homeostasis. This surveillance is accomplished by PMo crawling on the apical side of the endothelium through regulated interactions of integrins and chemokine receptors with their endothelial ligands. We propose that the search mode governs the intravascular motility of PMo in vivo in a similar way to T cells looking for antigen in tissues. Signs of damage to the luminal side of the endothelium (local death, oxidized LDL, amyloid deposits, tumor cells, pathogens, abnormal red cells, etc.) will change the diffusive random towards a Lèvy-like crawling enhancing their recognition and clearance by PMo damage receptors as the integrin αMβ2 and CD36. This new perspective can help identify new actors to promote unique PMo intravascular actions aimed at maintaining endothelial fitness and combating harmful microparticles involved in diseases as lung metastasis, Alzheimer’s angiopathy, vaso-occlusive disorders, and sepsis.

129 Stimulating the Cholinergic Anti-inflammatory Pathway Alters Inflammatory Cell Mobilization after Burn Injury

Introduction Severe burn injury causes a systemic inflammatory response (SIRS) that is characterized by mobilization of inflammatory cells into the circulation and is associated with distant organ injury that can lead to significant morbidity and mortality. The cholinergic anti-inflammatory pathway, mediated by the vagus nerve, regulates the anti-inflammatory response to injury and infection. We have previously shown in models of burn injury that stimulating the vagus nerve may be a potential therapy aimed at limiting SIRS. Here, we hypothesized that stimulating the vagus nerve (VNS) would limit the SIRS response by altering the mobilization and trafficking of inflammatory cells after burn injury. Methods Wild type 10–12-week-old C57BL/6 mice were injured with a 30% total body surface area steam burn. A separate cohort of animals was treated with electrical stimulation of the cervical vagus nerve for 10 minutes immediately post-burn. Bone marrow, blood and lung tissue were collected 24 hours after burn injury. Flow cytometry of bone marrow was performed to measure Lineage- c-kit± Sca-1+ (LSK) hematopoietic stem cells (HSC), then further analyzed to quantify changes in Long-term (LT) HSC, short-term (ST) HSC, and Multipotential Progenitor (MPP) compartments. Bone marrow, blood and perfused lung tissue were analyzed by flow cytometry using a panel of myeloid cell markers. Results Severe burn injury decreased bone marrow LSK expression by 50% compared to sham, with LT-HSC and MPP expression decreasing to a greater degree than ST-HSCs. VNS did not alter burn-induced changes in any bone marrow HSC cell type. Burn injury was associated with increased mobilization of CD45+CD11b+ monocytes and CD11b+Ly6Chi inflammatory monocytes into the peripheral blood and lung, while increased CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils was seen in the lung only. VNS significantly prevented the burn-induced increase in CD45+ inflammatory cells, CD11b+Ly6Clo patrolling monocytes and Gr1+Ly6C- neutrophils in the lung (see Figure), reducing their expression to sham levels, despite only modest changes to myeloid cell expression in the blood. Conclusions VNS attenuates myeloid cell cell trafficking to the lung after severe burn injury despite having no effect on emergency myelopoiesis in the bone marrow. Further studies are needed to define the mechanism by which the cholinergic anti-inflammatory pathway attenuates the SIRS response to burn.

Migrating Lung Monocytes Internalize and Inhibit Growth of Aspergillus fumigatus Conidia

Monocytes are important players to combat the ubiquitously present fungus Aspergillus fumigatus. Recruitment of monocytes to sites of fungal A. fumigatus infection has been shown in vivo. Upon exposure to A. fumigatus in vitro, purified murine and human blood monocytes secrete inflammatory cytokines and fungicidal mediators. Mononuclear tissue phagocytes are phenotypically and functionally different from those circulating in the blood and their role in antifungal defenses is much less understood. In this study, we identified a population of migrating CD43+ monocytes in cells isolated from rat distal lungs. These cells are phenotypically different from alveolar macrophages and show distinct locomotory behavior on the surface of primary alveolar cells resembling previously described endothelial patrolling monocytes. Upon challenge, the CD43+ monocytes internalized A. fumigatus conidia resulting in inhibition of their germination and hyphal growth. Thus, migrating lung monocytes might play an important role in local defense against pulmonary pathogens.

Relationship between dynamic changes in subpopulations of blood monocytes and the development of complications in patients with acute myocardial infarction

The aim – to determine the extent of different subpopulations of blood monocytes in acute myocardial infarction (AMI) with ST-segment elevation patients on day 1 and 7 and to evaluate the relationship between their content and the dynamics of changes and the risk of complications after AMI.Materials and methods. The composition of individual subpopulations of monocytes in the peripheral venous blood (and general clinical and biochemical blood tests) was evaluated in 50 pts with STEMI (who were admitted within 6 hours after the onset of the disease) at admission (before primary PCI) and on day 7. All patients received standard recommended therapy. Dynamic heart echocardiography was also performed on the 1st and 7th day. All patients were divided into 2 groups depending on the dynamical increase (1 group – 21 pts) or decrease (2 group – 29 pts) of classical monocytes (CD14hiCD16–) subpopulation during 7 days of follow-up. The control group included 15 healthy subjects with no signs of coronary heart disease and 23 pts with chronic coronary heart disease without AMI.Results and discussion. In subgroup 1, the percentage of the «classical» fraction of monocytes during the observation increased to 89.0±1.2 %, which was 4.2 % more than on the 1st day and 12.5 % more than in the control group (р<0.05), while the absolute amount of classic monocytes on day 7 increased by 48 % compared to initial value (р<0.01). The percentage of «intermediate» (CD14hiCD16+) blood monocytes in patients of this subgroup on the 1st day of hospitalization was 70 % higher than in the control group, and 42 % higher than in the 2nd subgroup of patients (р<0,001), however, on the 7th day it decreased by 30 % compared to baseline, although it remained by 8 % more than in the control group (the absolute number of «intermediate» monocytes did not change). The activation index (IA) of the «intermediate» monocytes on the first day did not differ between subgroups and was 40 % higher than in the control group (р<0.001). However, in the dynamics of observation, in patients of subgroup 1, this figure did not change, while in subgroup 2 IA decreased by 60 % (р<0.001). Despite the fact that the absolute number of anti-inflammatory («patrolling») (CD14+lowCD16++) monocytes did not change until the 7th day of observation (and their percentage decreased slightly), their IA was significantly lower than in the control group (95 %) and in patients of subgroup 2 (92 %, р<0,001). In patients of subgroup 2, the decrease of the percentage of «classic» monocytes was –7.7 % (from 90.4±0.8 to 83.4±1.2 %). Despite the fact that the number and percentage of intermediate monocytes increased in dynamics, their IA decreased almost 2 times, which may indicate a decrease in the pro-inflammatory ability these monocytes. The percentage and number of «patrolling» monocytes increased in dynamics by 37.4 % (р<0.0001) and by 268.3 % (р<0.01), respectively. IA of patrolling monocytes was almost 12 and 7 times higher than in patients of subgroup 1 on the 1st and 7th day of observation, respectively, which may indicate a significant activation of anti-inflammatory activity of patrolling monocytes. Intracardiac thrombosis was 3.3 times more common in patients of subgroup 1, in this subgroup was also more often noted (compared to the subgroup 2): dilatation of the left ventricle (almost 8 times), reduction of left ventricular ejection fraction (4 times), and pathological post-infarction remodeling of the left ventricle (almost 7 times).Conclusions. The results of the study indicate the important role of different subpopulations of blood monocytes in the processes of myocardial damage and recovery (in particular, the pro-inflammatory role of increasing the number of classical monocytes and increasing the activity of intermediate monocytes, as well as the anti-inflammatory role of increasing the number, percentage and activity of patrolling monocytes) in patients with AMI and can be the basis for developing new approaches to the diagnosis and prevention of complications of this disease.

Patrolling monocytes are recruited and activated by diabetes to protect retinal microvessels

In diabetes there is a long latency between onset of hyperglycemia and appearance of structural microangiopathy. Because Ly6C^low patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. <p>We found that, in wild-type mice,<b> </b>diabetes reduced PMo in the general circulation but increased by 4-fold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1^-/- mice ─ a model of absence of PMo ─ there was no increase in leukostasis at all; and at 6 months of diabetes the number of retinal acellular capillaries almost doubled when compared to diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculo-protective, and housekeeping activities; as well as profound suppression of genes related to inflammation and apoptosis. Pro-migratory CXCR4 was no longer upregulated at longer duration, when retinal acellular capillaries begin to increase.</p> <p>Thus, after short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes, and document within the natural history of diabetic retinopathy processes of protection-repair that can provide novel paradigms for prevention. </p>

Patrolling monocytes are recruited and activated by diabetes to protect retinal microvessels

In diabetes there is a long latency between onset of hyperglycemia and appearance of structural microangiopathy. Because Ly6C^low patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. <p>We found that, in wild-type mice,<b> </b>diabetes reduced PMo in the general circulation but increased by 4-fold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1^-/- mice ─ a model of absence of PMo ─ there was no increase in leukostasis at all; and at 6 months of diabetes the number of retinal acellular capillaries almost doubled when compared to diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculo-protective, and housekeeping activities; as well as profound suppression of genes related to inflammation and apoptosis. Pro-migratory CXCR4 was no longer upregulated at longer duration, when retinal acellular capillaries begin to increase.</p> <p>Thus, after short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes, and document within the natural history of diabetic retinopathy processes of protection-repair that can provide novel paradigms for prevention. </p>