Ly6Chi Inflammatory Monocytes Research Articles

Abstract WP317: Phagocytic Potentials of Purinergic Receptor P2x4 Blockade After Ischemic Stroke

Backgrounds: We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides neuroprotection by reducing acute excessive inflammation and thus can be potential drug targets to treat ischemic stroke. However, the mechanism how P2X4R blockade provide long term benefits are not known. Here we hypothesize that P2X4R blockade increase phagocytic uptake of damaged or dead tissue and facilitate swift recovery. Methods: We used both 1μm size fluorescent phagocytic bead (Polyscience Inc) and pHrodo Red zymosan A Bioparticles conjugate (Thermofisher) to study in vitro and ex-vivo phagocytic uptake by LPS (200 μg/ml for 2 hours) primed Bone Marrow Derived Macrophages (BMDMs) from P2X4R KO and littermate WT mice. We also used flow sorted Ly6C hi and low monocytes for phagocytic uptake study obtained from perilesional ipsilateral mouse brain tissue of P2X4R KO and littermate WT mice after 3 or 7 days of stroke. The later mice were subjected to 60 mins of transient middle cerebral artery occlusion. Results: Both phagocytic beads and pHrodo bioparticle uptake study suggest significantly higher uptake (1.5 and 2-fold P<0.05 vs WT student t test n=4 mice/group x 2 technical replication) by BMDMs obtained from P2X4R KO mice. FACS sorted infiltrated Ly6Chi inflammatory monocytes from global P2X4R KO mice brain after 3 days of stroke show significantly (*P>0.05 vs WT) high phagocytic bead uptake as compared to WT control. Further these monocytes also showed elevated expression of CD36, a marker for scavenger receptor at 7 days after stroke. Conclusions: This study suggest that P2X4R blockade provide swift and sustained neuroprotection by increasing phagocytic uptake of damage tissue after ischemic stroke.

Abstract A012: STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity

Abstract The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is triggered by the sensing of cytosolic DNA, triggering the production of type I interferon (IFN-I) and play a crucial role in host defense against pathogens. This pathway also induces an anti-tumor response when tumor-derived DNA is sensed. Small molecule agonists called STING agonists activate this pathway and have been shown to cause tumor regression in mice. However, it is unclear how immune cells respond to STING agonist-induced IFN-I. In our study, we observed that monocytes were abundantly recruited to tumors during STING agonist therapy, and the lack of CCR2+ Ly6Chi inflammatory monocytes in CCR2-knockout (KO) mice led to impaired responses to STING agonist therapy of B16F10 and MC-38 tumors. Single-cell RNA sequencing (scRNA-Seq) of CD45+ cells from lymph nodes and tumors of Ifnar1 mixed bone marrow chimeric mice in which half of the immune cells lacked IFN-I receptors revealed that inflammatory monocytes upregulated chemokine receptor ccr5 and genes associated with MHC-I restricted antigen presentation in response to IFN-I. Using mice with B16.OVA.Tomato red fluorescent tumors, we demonstrated that IFN-I-responsive monocytes captured fluorescent antigens in tumors and transported them to lymph nodes. Additionally, the administration of CCR5 antagonist, maraviroc, significantly reduced the number of fluorescent antigen-carrying monocytes in the lymph nodes of red fluorescent tumor-bearing mice treated with STING agonists, indicating the involvement of CCR5 in monocyte-mediated antigen transport. Furthermore, NicheNet analysis of scRNA-Seq data showed that IL-18 - IL-18R1 interaction was the top predicted interaction between inflammatory monocytes and CD8+ T cells or NK cells. Blocking IL-18 remarkedly reduced the therapeutic efficacy of STING agonist treatment. Moreover, IFN-I acted on monocytes to upregulate glucocorticoid-induced TNFR-related (GITRL) providing a pro-survival signal to T cells. Deletion of GITRL on CCR2+ monocytes led to impaired tumor control, and the absence of the cognate receptor GITR on T cells led to impaired anti-tumor effector T cell responses in the tumors treated with and tissue-resident memory T cell accumulation in the skins that rejected tumors with STING agonist therapy. Taken together, our data suggests that STING agonists induce IFN-I-responsive monocytes to promote anti-tumor responses through secreting IL-18, potentially through transporting antigens to lymph nodes, and through providing a pro-survival signal for T cell accumulation in the tumors during STING agonist therapy. Citation Format: Tianning Yu, Melanie Girard, Tania H Watts. STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A012.

MyD88 in myeloid cells promotes angiotensin II-induced vascular inflammation and is associated with all-cause mortality and incident heart failure in humans with arterial hypertension

Abstract Background Angiotensin II (AngII) causes hypertension and promotes vascular accumulation of inflammatory cells. We aimed to determine how myeloid factor of differentiation 88 (MyD88) contributes to vascular dysfunction and arterial hypertension in mice and humans. Methods Male C57BL/6 as well as MyD88-/-, LysMCre/wtMyD88LSL/LSL, LysMCre/wt, TLR2-/-, TLR4-/-, TLR7-/- and TLR9-/- were investigated in the AngII-model of hypertension (1 mg/kg/d for 7days). Biodata analyses were performed in the Gutenberg-Health Study (GHS), a population based cohort. In addition, we performed computational analyses of known hypertension-related genes from genome-wide association studies (GWAS) to investigate whether they preferentially interact with MyD88 in the human protein network. Results MyD88 deficiency attenuated AngII-induced blood pressure increase and endothelial dysfunction in conductance and resistance vessels. Vascular mRNA expression levels of vcam-1, nos2, nox2 were decreased in AngII-infused MyD88-/- mice compared to C57BL/6 controls. Flow cytometric analyses revealed that AngII-induced aortic accumulation of CD11b+Ly6Chi inflammatory monocytes was significantly dampened in MyD88-/- mice. AngII increased mRNA expression of cd62L, cd68 and ccl2, which was blocked in MyD88-/- mice, indicating a role of MyD88 for myeloid cell activation and differentiation. Additionally, aortic interferon-g+ NK cells and mRNA levels of interleukin-12 and interleukin-1b were reduced in AngII-treated MyD88-/- mice. Bone marrow transfer experiments demonstrated a vasoprotective effect of MyD88 deficiency in bone marrow-derived cells, and selective expression of MyD88 in LysMCre/wtMyD88LSL/LSL mice restored AngII-induced pathology, revealing that myelomonocytic cells drives vascular dysfunction in a MyD88-dependent manner. Furthermore, data from 1,274 individuals in the GHS indicated, that MyD88 mRNA expression was associated with all cause mortality and incident chronic heart failure after a median follow-up of 11.6 years. Computational analysis of the human protein interaction network demonstrated that MyD88 is significantly connected to proteins encoded by genetic loci associated with blood pressure traits in multiple GWAS. Conclusion We provide evidence, that MyD88 expressed by myelomonocytic cells promotes AngII-induced vascular dysfunction and arterial hypertension. MyD88 might be used as an inflammatory diagnostic marker for the risk of death in hypertensive patients.

Differential skin immune responses in mice intradermally infected with Candida auris and Candida albicans.

Candida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue, resulting in outbreaks of nosocomial infections. Understanding the factors that regulate C. auris skin colonization is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We established a model of intradermal C. auris inoculation in mice and found that mice infected with C. auris elicit less potent innate and adaptive immune responses in the infected skin compared to C. albicans. These findings help explain the clinical observation of persistent C. auris colonization in skin tissue.

Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS.

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

B cell-activating factor (BAFF) from dendritic cells, monocytes and neutrophils is required for B cell maturation and autoantibody production in SLE-like autoimmune disease.

B cell-activating factor (BAFF) contributes to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Although several anti-BAFF Abs and derivatives have been developed for the treatment of SLE, the specific sources of BAFF that sustain autoantibody (auto-Ab) producing cells have not been definitively identified. Using BAFF-RFP reporter mice, we identified major changes in BAFF-producing cells in two mouse spontaneous lupus models (Tlr7 Tg mice and Sle1), and in a pristane-induced lupus (PIL) model. First, we confirmed that similar to their wildtype Tlr7 Tg and Sle1 mice counterparts, BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice had increased BAFF serum levels, which correlated with increases in plasma cells and auto-Ab production. Next, using the RFP reporter, we defined which cells had dysregulated BAFF production. BAFF-producing neutrophils (Nphs), monocytes (MOs), cDCs, T cells and B cells were all expanded in the spleens of BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice compared to controls. Furthermore, Ly6Chi inflammatory MOs and T cells had significantly increased BAFF expression per cell in both spontaneous lupus models, while CD8- DCs up-regulated BAFF expression only in the Tlr7 Tg mice. Similarly, pristane injection of BAFF-RFP mice induced increases in serum BAFF levels, auto-Abs, and the expansion of BAFF-producing Nphs, MOs, and DCs in both the spleen and peritoneal cavity. BAFF expression in MOs and DCs, in contrast to BAFF from Nphs, was required to maintain homeostatic and pristane-induced systemic BAFF levels and to sustain mature B cell pools in spleens and BMs. Although acting through different mechanisms, Nph, MO and DC sources of BAFF were each required for the development of auto-Abs in PIL mice. Our findings underscore the importance of considering the relative roles of specific myeloid BAFF sources and B cell niches when developing treatments for SLE and other BAFF-associated autoimmune diseases.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

CD146+ Umbilical Cord Mesenchymal Stem Cells Exhibit High Immunomodulatory Activity and Therapeutic Efficacy in Septic Mice.

Several studies have shown that MSCs can significantly improve the survival of sepsis animals. CD146+ mesenchymal stem cells (MSCs) correlate with high therapeutic potency. However, their therapeutic effect on sepsis and detail mechanisms have not been explored. The effect of CD146±MSCs on differentiation of Treg, Th1, Th17 subsets was evaluated by flow cytometry. The effects of CD146±MSCs on RAW264.7 phagocytosis and LPS-stimulated polarization were studied using a co-culture protocol. Luminex bead array and RNA sequencing were employed to determine the mechanisms of MSCs on LPS-stimulated RAW264.7. The Arg1 protein was detected by Western blot. CD146±MSCs were injected into LPS-induced sepsis mice by tail vein. The therapeutic effect was assessed by organ HE staining, T-cell subsets, cytokine in plasma, peritoneal macrophages, infiltrating monocytes subpopulations. In vitro, CD146+MSCs could significantly increase the proportion of Treg cells. Co-culture with CD146+MSCs increase the phagocytic rate of RAW264.7. CD146+MSCs regulate M2-type macrophages production more rapidly. The transcript profile differences between the CD146+MSCs and CD146-MSCs groups were clustered in arginine metabolism pathways. CD146+MSCs decreased NO production and increased ARG1 expression. CD146+MSCs secreted higher level of IL15,IFNγ, VEGF and lower level of IL1β, IL8 under LPS stimuli. In vivo, The level of IL10 at 24h and CXCL1, IFNγ at 12h in CD146+MSCs group was the highest. CD146+MSCs treatment enhances the phagocytic capacity of peritoneal macrophages. CD146+MSCs also increases the ratios of CD11b+Ly6Clo reparative monocytes and CD11b+Ly6Chi inflammatory monocytes until 24h. Compared with CD146-MSCs, CD146+MSCs can accelerate the end of the inflammatory response and have robust anti-inflammatory effects, by increasing the Treg cells, promoting macrophage phagocytosis, enhancing the reparative macrophage, secreting more VEGF, etc.

ASGR1 promotes liver injury in sepsis by modulating monocyte-to-macrophage differentiation via NF-κB/ATF5 pathway

AimsLiver is a pivotal organ for sepsis-induced injury and approximately 40 % of liver injury results from sepsis. During hepatic injury, monocyte-to-macrophage differentiation is a key event because it results in the regulation of immune response. Asialoglycoprotein receptor 1 (ASGR1) is enriched in classical monocyte of peripheral blood mononuclear cells (PBMCs). We aimed to explore the effect of ASGR1 on monocyte-to-macrophage differentiation and the modulation of sepsis-induced liver injury. Main methodsASGR1-knockdown/overexpression THP-1 cells and mice bone marrow-derived macrophages (BMDMs) induced by PMA and 30 % L929-cell conditioned medium were utilized to test the impact of ASGR1 on monocyte-to-macrophage differentiation and molecular mechanism respectively. Expression of differentiation specific factors were assessed via flow cytometry and real-time quantitative PCR. RNA-sequencing (RNA-seq) analysis revealed the effect of ASGR1 on monocyte-to-macrophage differentiation. Further, differentiation specific factors ATF5 and NF-κB pathways were examined via Western blot. The interaction between ASGR1 and ATF5 was further examined by co-IP. Finally, LPS-induced ASGR1-knockdown mice sepsis was used to investigate the effect of ASGR1 on monocyte-to-macrophage differentiation, liver injury and survival. Key findingsASGR1 promoted monocyte-to-macrophage differentiation via up-regulating CD68, F4/80 and CD86. Additionally, inhibited-ASGR1 decreased ATF5 expression by suppressing phosphorylation of NF-κB and IKBa in vitro and in vivo. ASGR1-knockdown mice suppressed Ly6Chi inflammatory monocytes in PBMCs, and restrained CD45+CD11bhiF4/80+Ly6Clo monocyte-derived macrophages and CD45+CD11b+F4/80+Ly6C+ inflammatory macrophages in livers. It also suppressed the level of IL-1β, IL-6, TNF-α and alleviated liver injury and improved survival after sepsis. SignificanceASGR1 is a negative regulator for sepsis-induced liver injury and survival.

Multidimensional immunoimaging to characterize the inflammatory reaction after permanent and temporary coronary artery occlusion in mice

Abstract Background The immune response following acute myocardial infarction encompasses a delicate balance between inflammatory and reparative programs. Our knowledge of these complex mechanisms is mainly derived from studies using a murine model of permanent coronary artery occlusion. In this study we developed, validated and implemented multiparametric imaging methods to investigate cardiac function and the systemic immune response in transient or permanent coronary artery occlusion mouse models. Methods The myocardial infarction models encompassed either transient (40 min) or permanent LAD occlusion in C57BL/6 mice, and non-infarcted mice were used as controls. Two or seven days later, the animals subjected to systemic immunoimaging of the bone marrow, spleen and myocardium with late gadolinium enhancement cardiac MRI (LGE cMRI), 18F-fluorodeoxyglucose (18F-FDG) PET, 18F-Fluorothymidine (18F-FLT) PET, 64Cu-CCR2 PET targeting inflammatory monocytes, 89Zr-CD11b nanobody PET and 19F-HDL-PERFECTA MRI, both targeting myeloid cells. In addition, the same myocardial infarction models were applied to atherosclerosis-prone Apoe−/− and systemic inflammation and plaque progression were assessed by flow cytometry and immunohistochemistry four weeks after infarction. Results Through LGE cMRI and 18F-FDG PET, we observed that temporary coronary occlusion resulted in a smaller infarct size, better cardiac function and viability compared to permanent occlusion. Multiparametric immunoimaging targeting CD11b+ cells by 89Zr-CD11b nanobody PET and Ly6Chi inflammatory monocytes by 64Cu-CCR2 PET demonstrated that mice subjected to transient coronary occlusion had less immune cell influx to the ischemic myocardium. This finding was confirmed by flow cytometry analysis of the infarct zone. In contrast, both myocardial infarction models cause a similar systemic immune response in the bone marrow and spleen as observed with multimodal imaging with subsequent similar numbers of CD11b+ cells in the blood. Both permanent and temporary coronary artery occlusion aggravate atherosclerosis in Apoe−/− mice with higher macrophage and Ly6Chi monocyte numbers in aortas and larger plaque size compared to Apoe−/− mice without myocardial infarction. Conclusions We developed and employed multimodal, multiparametric imaging protocols to characterize the immune response in the heart, bone marrow and spleen in two models of myocardial infarction. While cardiac function was superior in the ischemia reperfusion model, both types of myocardial infarction accelerated atherosclerosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): NIH and DFG

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Liposome-encapsulated curcumin attenuates HMGB1-mediated hepatic inflammation and fibrosis in a murine model of Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism with predominant hepatic manifestations. Left untreated, it can be fatal. Current therapies focus on treating copper overload rather than targeting the pathophysiology of copper-induced liver injuries. We sought to investigate whether liposome-encapsulated curcumin (LEC) could attenuate the underlying pathophysiology of WD in a mouse model of WD. Subcutaneous administration in a WD mouse model with ATP7B knockout (Atp7b-/-) resulted in robust delivery of LEC to the liver as determined by in-vitro and in-vivo imaging. Treatment with LEC attenuated hepatic injuries, restored lipid metabolism and decreased hepatic inflammation and fibrosis, and thus hepatosplenomegaly in Atp7b-/- mice. Mechanistically, LEC decreased hepatic immune cell and macrophage infiltration and attenuated the hepatic up-regulation of p65 by preventing cellular translocation of high-mobility group box-1 (HMGB-1). Moreover, decreased translocation of HMGB1 was associated with reduced splenic CD11b+/CD43+/Ly6CHi inflammatory monocyte expansion and circulating level of proinflammatory cytokines. Nevertheless there was no change in expression of oxidative stress-related genes or significant copper chelation effect of LEC in Atp7b-/- mice. Our results indicate that treatment with subcutaneous LEC can attenuate copper-induced liver injury in an animal model of WD via suppression of HMGB1-mediated hepatic and systemic inflammation. These findings provide important proof-of-principle data to develop LEC as a novel therapy for WD as well as other inflammatory liver diseases.

STK25 acts as a regulator of leukocyte migration

Abstract The recruitment of leukocytes to sites of infection is an essential component of the innate immune response to pathogens. Defects in leukocyte migration can render the host susceptible to recurrent infections. In our efforts to identify new mediators of innate immunity, we found that serine/threonine protein kinase 25 (STK25) promotes TLR-induced proinflammatory cytokine production. While STK25 has previously been implicated in the regulation of cell migration, its role in leukocyte trafficking has not been defined. To investigate the involvement of STK25 in leukocyte migration, we evaluated baseline hematological parameters from Stk25-deficient (Stk25−/−) mice via a Hemavet. We found that Stk25−/− mice exhibit reduced circulating lymphocytes, neutrophils, and monocytes at steady-state compared to age- and gender-matched wild-type littermate (WT) mice. Immunophenotyping by flow cytometry confirmed these findings and revealed a significant reduction in the frequency of Ly6Chi inflammatory monocytes in Stk25−/− peripheral blood (PB) relative to WT. In contrast, we observed no apparent change in the frequencies of neutrophils and Ly6Chi inflammatory monocytes in Stk25−/− bone marrow, suggesting a defect in leukocyte migration. Finally, we examined the role of STK25 in neutrophil trafficking via an in vitro chemotaxis assay. We found that PB neutrophils from Stk25−/− mice displayed a defect in migration toward WT serum, suggesting a cell-intrinsic role for STK25 in the control of cell trafficking. Additionally, PB neutrophils from WT mice exhibited reduced migration toward Stk25−/− serum, supporting a cell-extrinsic function for STK25. Thus, our data support a new role for STK25 as a mediator of leukocyte trafficking.

Inflammatory Monocyte Counts Determine Venous Blood Clot Formation and Resolution.

Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation, clot inflammation, and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1, Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation, similarly suspends clotting, while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation, growth, and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages.

The Autoimmune Risk R262W Variant of the Adaptor SH2B3 Improves Survival in Sepsis.

The single-nucleotide polymorphism (SNP) rs3184504 is broadly associated with increased risk for multiple autoimmune and cardiovascular diseases. Although the allele is uniquely enriched in European descent, the mechanism for the widespread selective sweep is not clear. In this study, we find the rs3184504*T allele had a strong association with reduced mortality in a human sepsis cohort. The rs3184504*T allele associates with a loss-of-function amino acid change (p.R262W) in the adaptor protein SH2B3, a likely causal variant. To better understand the role of SH2B3 in sepsis, we used mouse modeling and challenged SH2B3-deficient mice with a polymicrobial cecal-ligation puncture (CLP) procedure. We found SH2B3 deficiency improved survival and morbidity with less organ damage and earlier bacterial clearance compared with control mice. The peritoneal infiltrating cells exhibited augmented phagocytosis in Sh2b3 -/- mice with enriched recruitment of Ly6Chi inflammatory monocytes despite equivalent or reduced chemokine expression. Rapid cycling of monocytes and progenitors occurred uniquely in the Sh2b3 -/- mice following CLP, suggesting augmented myelopoiesis. To model the hypomorphic autoimmune risk allele, we created a novel knockin mouse harboring a similar point mutation in the murine pleckstrin homology domain of SH2B3. At baseline, phenotypic changes suggested a hypomorphic allele. In the CLP model, homozygous knockin mice displayed improved mortality and morbidity compared with wild-type or heterozygous mice. Collectively, these data suggest that hypomorphic SH2B3 improves the sepsis response and that balancing selection likely contributed to the relative frequency of the autoimmune risk variant.

Inflammatory Monocytes and Subsets of Macrophages with Distinct Surface Phenotype Correlate with Specific Integrin Expression Profile during Murine Sepsis.

Monocytes and macrophages participate in both pro- and anti-inflammatory responses during sepsis. Integrins are the cell adhesion receptors that mediate leukocyte migration and functions. To date, it is not known whether integrin profiles correlate with their trafficking, differentiation, and polarization during sepsis. In this study, using endotoxemia and cecal ligation and puncture model of murine sepsis, we have analyzed the role of surface integrins in tissue-specific infiltration, distribution of monocytes and macrophages, and their association with inflammation-induced phenotypic and functional alterations postinduction (p.i.) of sepsis. Our data show that Ly-6Chi inflammatory monocytes infiltrated into the peritoneum from blood and bone marrow within a few hours p.i. of sepsis, with differential distribution of small (Ly-6CloCD11bloF4/80lo) and large peritoneal macrophages (Ly-6CloCD11bhiF4/80hi) in both models. The results from flow cytometry studies demonstrated a higher expression of integrin α4β1 on the Ly-6Chi monocytes in different tissues, whereas macrophages in the peritoneum and lungs expressed higher levels of integrin α5β1 and αvβ3 in both models. Additionally, F4/80+ cells with CD206hiMHCIIlo phenotype increased in the lungs of both models by six hours p.i. and expressed higher levels of integrin αvβ3 in both lungs and peritoneum. The presence of such cells correlated with higher levels of IL-10 and lower levels of IL-6 and IL-1β transcripts within six hours p.i. in the lungs compared with the mesentery. Furthermore, bioinformatic analysis with its experimental validation revealed an association of integrin α4 and α5 with inflammatory (e.g., p-SRC) and integrin αv with regulatory molecules (e.g., TGFBR1) in macrophages during sepsis.

CXCL12/CXCR4 Mediates Orthodontic Root Resorption via Regulating the M1/M2 Ratio

Mechanical force–induced external root resorption is a major clinical side effect of orthodontic treatment. Recent work has revealed that M1 macrophages play a vital role in promoting orthodontic root resorption (ORR), but the mechanism of how mechanical force stimulation increases the M1/M2 macrophage ratio in periodontal tissue is poorly understood. In the current study, we showed that C-X-C motif chemokine 12 (CXCL12)+ periodontal ligament cells (PDLCs) and C-X-C chemokine receptor type 4 (CXCR4)+ monocytes in the periodontal ligament (PDL) were significantly increased after force application with ongoing root resorption, and these effects were partially rescued after force removal in mice. The expression of CXCL12 in PDLCs was increased by force stimulation in a time- and intensity-dependent manner in vitro. Blockage of the CXCL12/CXCR4 axis using CXCR4 antagonist AMD3100 was sufficient to alleviate ORR and reverse the force-enhanced M1/M2 macrophage ratio. Further mechanism exploration showed that Ly6Chi inflammatory monocytes homed in a CXCL12/CXCR4 axis-dependent manner. The number and proportion of CD11b+ Ly6Chi inflammatory monocytes in cervical lymph nodes were significantly increased by force loading, accompanied by decreased CD11b+ Ly6Chi monocytes in the blood. These changes were blunted by intraperitoneal injection of AMD3100. In addition, blockage of the CXCL12/CXCR4 axis effectively reversed M2 suppression and promoted M1 polarization. Collectively, results indicate that force-induced CXCL12/CXCR4 axis mediates ORR by increasing the M1/M2 ratio in periodontal tissues through attracting Ly6Chi inflammatory monocytes and modulating macrophage polarization. The results also imply that AMD3100 is potentially inhibitory to root resorption.

Antibiotic-induced gut dysbiosis leads to activation of microglia and impairment of cholinergic gamma oscillations in the hippocampus

Antibiotics are widely applied for the treatment of bacterial infections, but their long-term use may lead to gut flora dysbiosis and detrimental effects on brain physiology, behavior as well as cognitive performance. Still, a striking lack of knowledge exists concerning electrophysiological correlates of antibiotic-induced changes in gut microbiota and behavior. Here, we investigated changes in the synaptic transmission and plasticity together with behaviorally-relevant network activities from the hippocampus of antibiotic-treated mice. Prolonged antibiotic treatment led to a reduction of myeloid cell pools in bone marrow, circulation and those surveilling the brain. Circulating Ly6Chi inflammatory monocytes adopted a proinflammatory phenotype with increased expression of CD40 and MHC II. In the central nervous system, microglia displayed a subtle activated phenotype with elevated CD40 and MHC II expression, increased IL-6 and TNF production as well as with an increased number of Iba1 + cells in the hippocampal CA3 and CA1 subregions. Concomitantly, we detected a substantial reduction in the synaptic transmission in the hippocampal CA1 after antibiotic treatment. In line, carbachol-induced cholinergic gamma oscillation were reduced upon antibiotic treatment while the incidence of hippocampal sharp waves was elevated. These alterations were associated with the global changes in the expression of neurotrophin nerve growth factor and inducible nitric oxide synthase, both of which have been shown to influence cholinergic system in the hippocampus. Overall, our study demonstrates that antibiotic-induced dysbiosis of the gut microbiome and subsequent alteration of the immune cell function are associated with reduced synaptic transmission and gamma oscillations in the hippocampus, a brain region that is critically involved in mediation of innate and cognitive behavior.

Interferon-γ-Producing CD4+ T Cells Drive Monocyte Activation in the Bone Marrow During Experimental Leishmania donovani Infection.

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2 -/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10 -/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10 -/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.