Leukocyte Deficiency Research Articles

Epilepsy and Cognition in GM2-Gangliosidosis B1 Variant – Experience of a Tertiary Hospital

Introduction: GM2 gangliosidosis B1 variant (GM2B1) is an autosomal recessive disorder due to deficiency of β-hexosaminidase A, leading to the lysosomal storage of GM2 gangliosides in neuronal tissue and neuronal death. Symptoms include progressive motor coordination impairment and neurodegeneration, in children with previously normal development, leading to early death. Objective: Clinical characterization of patients with GM2B1 with a focus on epileptic manifestations. Methods: A descriptive retrospective study was conducted, analyzing clinical records of patients diagnosed with GM2B1 and followed at our Hospital Neuropediatric Department in the decade 2013-2022. Results: Four patients (three female) from three families diagnosed with GM2B1 were identified. The median age at diagnosis was 70 months. The most frequent symptoms at presentation were developmental regression (all children), language impairment (three), and epileptic seizures (two). Enzymatic deficiency in leukocytes and pathogenic variants in the HEXA gene were demonstrated in all cases. The pathogenic variant c.533G>A(p.R178H) in exon 5 was present in seven of the eight alleles. All patients experienced language impairment (Md=42 mos) with complete language loss (Md=78 mos). Loss of walking ability occurred in three patients (Md=96 mos). All patients experienced epileptic seizures during the course of the disease, with a median onset of seizures at 55 months. Initial seizures were classified as atypical absences (two), tonic seizures (one) and myoclonic seizures (one). Electroencephalographic evaluation revealed slow basal rhythm and focal paroxysmal activity in all cases. All were treated with antiseizure medication, two requiring a combination of three drugs. Conclusion: GM2B1 encompasses a wide clinical spectrum, with heterogenous age of symptom onset, clinical presentation and disease progression. Epilepsy is a common comorbidity in GM2B1, with variable seizure type and severity, and may be difficult to control. Timely diagnosis, coupled with multidisciplinary clinical follow-up, is crucial to improve quality of life of patients and enable genetic counseling.

Disruption of the Uty epigenetic regulator locus in hematopoietic cells phenocopies the profibrotic attributes of Y chromosome loss in heart failure.

Heart failure affects millions of people worldwide, with men exhibiting a higher incidence than women. Our previous work has shown that mosaic loss of the Y chromosome (LOY) in leukocytes is causally associated with an increased risk for heart failure. Here, we show that LOY macrophages from the failing hearts of humans with dilated cardiomyopathy exhibit widespread changes in gene expression that correlate with cardiac fibroblast activation. Moreover, we identify the ubiquitously transcribed t et ratricopeptide Y-linked (Uty) gene in leukocytes as a causal locus for an accelerated progression of heart failure in male mice with LOY. We demonstrate that Uty disruption leads to epigenetic alterations in both monocytes and macrophages, increasing the propensity of differentiation into profibrotic macrophages. Treatment with a transforming growth factor-β-neutralizing antibody prevented the cardiac pathology associated with Uty deficiency in leukocytes. These findings shed light on the mechanisms that contribute to the higher incidence of heart failure in men.

Retinitis pigmentosa and nanophthalmos in a patient with attenuated Hunter's syndrome.

To describe a case of retinitis pigmentosa and nanophthalmos in a patient with attenuated Hunter's syndrome. Fundus photography, total field electroretinogram, ultrasound, computerized visual field examination, biochemical examination and genetic testing were obtained. The fundus exam showed diffuse arteriolar attenuation, optic disc with regular contours, and pigment agglomerates like "bone spicules" in the middle periphery. Ultrasound examination revealed scleral thickening and short axial diameter in both eyes. The total field electroretinogram exam showed a subnormal result with greater impairment of the scotopic phase of the exam. Computerized visual field examination demonstrated a diffuse reduction in retinal sensitivity in the periphery. Biochemical examination showed increased urine glycosaminoglycan excretion and iduronate-2-sulphatase activity (IDS) deficiency in leukocytes, confirming the type II mucopolysaccharidosis. Molecular analysis revealed a novel missense mutation (p.A77D) in the IDS gene. The case report is about a patient presented an attenuated form of the syndrome, with no cognitive impairment. Ophthalmologic follow-up is still an important part of multidisciplinary treatment for Hunter's syndrome.

Morbidity and Mortality of Neutropenic Patients in Visceral Surgery: A Narrative Review

Leukocytes are essential for the function of the immune system and cell–cell interaction in the human body, but hematological diseases as well as chemotherapeutic treatments due to cancer lead to occasionally or even permanent leukocyte deficiency. Normally, more than 50% of leukocytes are neutrophilic granulocytes, and leukopenia is, therefore, mostly characterized by a decrease in neutrophilic granulocytes. The consequence of neutropenia is increased susceptibility to infection, but also healing disorders are suggestable due to the disturbed cell–cell interaction. While there is no surgical treatment for leucocyte disorders, patients suffering from neutropenia are sometimes in need of surgery for other reasons. Less is known about the morbidity and mortality of this patients, which is why this narrative review critically summarizes the results of recent research in this particular field. The results of this review suggest that neutropenic patients in need of emergency surgery have a higher mortality risk compared to non-neutropenic patients. In contrast, in elective surgery, there was not a clear tendency for a higher mortality risk of neutropenic patients. The role of neutrophilic granulocytes in inflammation and immunity in surgical patients is emphasized by the results, but most of the evaluated studies showed methodological flaws due to small sample sizes or risk of bias. Further research has to evaluate the risk for postoperative complications, particularly of infectious complications such as surgical site infections, in neutropenic patients undergoing elective surgery, and should address the role of neutrophilic function in postoperative morbidity and mortality.

Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice.

Melanocortin receptor 1 (MC1-R) is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbs cholesterol homeostasis, increases arterial leukocyte accumulation and accelerates atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. Since various cell types besides leukocytes express MC1-R, we aimed at investigating the specific contribution of leukocyte MC1-R to the development of atherosclerosis. For this purpose, male Apoe-/- mice were irradiated, received bone marrow from either female Apoe-/- mice or MC1-R deficient Apoe-/- mice (Apoe-/- Mc1re/e) and were analyzed for tissue leukocyte profiles and atherosclerotic plaque phenotype. Hematopoietic MC1-R deficiency significantly elevated total leukocyte counts in the blood, bone marrow and spleen, an effect that was amplified by feeding mice a cholesterol-rich diet. The increased leukocyte counts were largely attributable to expanded lymphocyte populations, particularly CD4+ T cells. Furthermore, the number of monocytes was elevated in Apoe-/- Mc1re/e chimeric mice and it paralleled an increase in hematopoietic stem cell count in the bone marrow. Despite robust leukocytosis, atherosclerotic plaque size and composition as well as arterial leukocyte counts were unaffected by MC1-R deficiency. To address this discrepancy, we performed an in vivo homing assay and found that MC1-R deficient CD4+ T cells and monocytes were preferentially entering the spleen rather than homing in peri-aortic lymph nodes. This was mechanistically associated with compromised chemokine receptor 5 (CCR5)-dependent migration of CD4+ T cells and a defect in the recycling capacity of CCR5. Finally, our data demonstrate for the first time that CD4+ T cells also express MC1-R. In conclusion, MC1-R regulates hematopoietic stem cell proliferation and tissue leukocyte counts but its deficiency in leukocytes impairs cell migration via a CCR5-dependent mechanism.

Oral Administration of Mavorixafor, a CXCR4 Antagonist, Increases Peripheral White Blood Cell Counts across Different Disease States

Oral Administration of Mavorixafor, a CXCR4 Antagonist, Increases Peripheral White Blood Cell Counts across Different Disease States

The effect of anti-cancer and anti-tuberculosis treatments in lung cancer patients with active tuberculosis: a retrospective analysis

BackgroundLung tuberculosis (TB) and lung cancer have a complex relationship. Data concerning TB treatment in lung cancer patients are still incomplete. The aim of this study was to investigate the effects of anti-cancer and anti-tuberculosis treatments in lung cancer patients with active lung TB.MethodsIn a retrospective cohort study, lung cancer patients with active lung TB were identified between January 2013 and December 2016. Age- and sex-matched lung cancer patients without tuberculosis were selected as control subjects. Anti-cancer and anti-tuberculosis treatments were administered according to the national guidelines. The clinical courses and responses of lung cancer patients with and without active lung TB were examined and compared.ResultsA total of 31 consecutive lung cancer patients were diagnosed with active lung TB. Fifty-one lung cancer patients without TB were enrolled as control subjects. Most patients in the two groups were elderly, had advanced non-small cell lung cancer and had tumor burdens. The anti-cancer treatment completion rate and response rate were not different between two group (87.1% in TB treatment patients vs. 92.2% in lung cancer patients, 77.4% in TB treatment patients vs. 88.2% in lung cancer patients, respectively). The anti-tuberculosis treatment completion rate and success rate was 87.1 and 80.7%. The median survival times were not different between two groups (52 weeks in TB treatment patients vs. 57 weeks in lung cancer patients). The change in Karnofsky performance score was also not different between two groups. The most common side effect in TB treatment patients was liver injury (61.3%). The most serious side effect in TB treatment patients was leukocyte deficiency (9.7% in Grade 3). Both of side effects mentioned above were not different between two groups.ConclusionBoth anti-cancer and anti-tuberculosis treatments can be safely and effectively administered in lung cancer patients with active lung TB. Attention should be paid to the risk of tuberculosis in lung cancer patients in TB high-burden countries.

A disintegrin and metalloproteinase domain-15 deficiency leads to exaggerated cigarette smoke-induced chronic obstructive pulmonary disease (COPD)-like disease in mice.

A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by cells implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its contributions to COPD are unknown. To address this gap, ADAM15 levels were measured in samples from cigarette smoke (CS)-versus air-exposed wild-type (WT) mice. CS-induced COPD-like disease was compared in CS-exposed WT, Adam15-/-, and Adam15 bone marrow chimeric mice. CS exposure increased Adam15 expression in lung macrophages and CD8+ T cells and to a lesser extent in airway epithelial cells in WT mice. CS-exposed Adam15-/- mice had greater emphysema, small airway fibrosis, and lung inflammation (macrophages and CD8+ T cells) than WT mice. Adam15 bone marrow chimera studies revealed that Adam15 deficiency in leukocytes led to exaggerated pulmonary inflammation and COPD-like disease in mice. Adam15 deficiency in CD8+ T cells was required for the exaggerated pulmonary inflammation and COPD-like disease in CS-exposed Adam15-/- mice (as assessed by genetically deleting CD8+ T cells in Adam15-/- mice). Adam15 deficiency increased pulmonary inflammation by rendering CD8+ T cells and macrophages resistant to CS-induced activation of the mitochondrial apoptosis pathway by preserving mTOR signaling and intracellular Mcl-1 levels in these cells. These results strongly link ADAM15 deficiency to the pathogenesis of COPD.

Intermittent rolling is a defect of the extravasation cascade caused by Myosin1e-deficiency in neutrophils

Neutrophil extravasation is a migratory event in response to inflammation that depends on cytoskeletal dynamics regulated by myosins. Myosin-1e (Myo1e) is a long-tailed class-I myosin that has not yet been studied in the context of neutrophil-endothelial interactions and neutrophil extravasation. Intravital microscopy of TNFα-inflamed cremaster muscles in Myo1e-deficient mice revealed that Myo1e is required for efficient neutrophil extravasation. Specifically, Myo1e deficiency caused increased rolling velocity, decreased firm adhesion, aberrant crawling, and strongly reduced transmigration. Interestingly, we observed a striking discontinuous rolling behavior termed "intermittent rolling," during which Myo1e-deficient neutrophils showed alternating rolling and jumping movements. Surprisingly, chimeric mice revealed that these effects were due to Myo1e deficiency in leukocytes. Vascular permeability was not significantly altered in Myo1e KO mice. Myo1e-deficient neutrophils showed diminished arrest, spreading, uropod formation, and chemotaxis due to defective actin polymerization and integrin activation. In conclusion, Myo1e critically regulates adhesive interactions of neutrophils with the vascular endothelium and neutrophil extravasation. Myo1e may therefore be an interesting target in chronic inflammatory diseases characterized by excessive neutrophil recruitment.

Bortezomib induces HSV-1 lethality in mice with neutrophil deficiency.

Bortezomib suppressing NF-κB activity is an effective therapy for patients with myeloma or lymphoma. However, this drug can cause adverse effects, neutropenia, and recurrent infections of herpes viruses. Among herpes viruses, HSV-1 can reactivate to induce mortality. The important issues regarding how bortezomib diminishes neutrophils, whether bortezomib can induce HSV-1 reactivation, and how bortezomib exacerbates HSV-1 infection, need investigation. Using the murine model, we found that bortezomib induced HSV-1 reactivation. Bortezomib diminished neutrophil numbers in organs of uninfected and HSV-1-infected mice and turned a nonlethal infection to lethal with elevated tissue viral loads. In vitro results showed that bortezomib and HSV-1 collaborated to enhance the death and apoptosis of mouse neutrophils. The leukocyte deficiency induced by chemotherapies is generally believed to be the cause for aggravating virus infections. Here we show the potential of pathogen to exacerbate chemotherapy-induced leukocyte deficiency.

A Disintegrin and Metalloproteinase Domain-8: A Novel Protective Proteinase in Chronic Obstructive Pulmonary Disease.

ADAM8 (a disintegrin and metalloproteinase domain-8) is expressed by leukocytes and epithelial cells in health, but its contribution to the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. To determine whether the expression of ADAM8 is increased in the lungs of patients with COPD and cigarette smoke (CS)-exposed mice, and whether ADAM8 promotes the development of COPD. ADAM8 levels were measured in lung, sputum, plasma, and/or BAL fluid samples from patients with COPD, smokers, and nonsmokers, and wild-type (WT) mice exposed to CS versus air. COPD-like lung pathologies were compared in CS-exposed WT versus Adam8-/- mice. ADAM8 immunostaining was reduced in macrophages, and alveolar and bronchial epithelial cells in the lungs of patients with COPD versus control subjects, and CS- versus air-exposed WT mice. ADAM8 levels were similar in plasma, sputum, and BAL fluid samples from patients with COPD and control subjects. CS-exposed Adam8-/- mice had greater airspace enlargement and airway mucus cell metaplasia than WT mice, but similar small airway fibrosis. CS-exposed Adam8-/- mice had higher lung macrophage counts, oxidative stress levels, and alveolar septal cell death rates, but lower alveolar septal cell proliferation rates and soluble epidermal growth factor receptor BAL fluid levels than WT mice. Adam8 deficiency increased lung inflammation by reducing CS-induced activation of the intrinsic apoptosis pathway in macrophages. Human ADAM8 proteolytically shed the epidermal growth factor receptor from bronchial epithelial cells to reduce mucin expression in vitro. Adam8 bone marrow chimera studies revealed that Adam8 deficiency in leukocytes and lung parenchymal cells contributed to the exaggerated COPD-like disease in Adam8-/- mice. Adam8 deficiency increases CS-induced lung inflammation, emphysema, and airway mucus cell metaplasia. Strategies that increase or prolong ADAM8's expression in the lung may have therapeutic efficacy in COPD.

Loss of the Rap1 effector RIAM results in leukocyte adhesion deficiency due to impaired β2 integrin function in mice

AbstractTalin is an integrin adaptor, which controls integrin activity in all hematopoietic cells. How intracellular signals promote talin binding to the integrin tail leading to integrin activation is still poorly understood, especially in leukocytes. In vitro studies identified an integrin activation complex whose formation is initiated by the interaction of active, guanosine triphosphate (GTP)-bound Ras-related protein 1 (Rap1) with the adapter protein Rap1-GTP–interacting adapter molecule (RIAM) followed by the recruitment of talin to the plasma membrane. Unexpectedly, loss-of-function studies in mice have shown that the talin-activating role of RIAM is neither required for development nor for integrin activation in platelets. In this study, we show that leukocyte integrin activation critically depends on RIAM both in vitro and in vivo. RIAM deficiency results in a loss of β2 integrin activation in multiple leukocyte populations, impaired leukocyte adhesion to inflamed vessels, and accumulation in the circulation. Surprisingly, however, the major leukocyte β1 integrin family member, α4β1, was only partially affected by RIAM deficiency in leukocytes. Thus, although talin is an essential, shared regulator of all integrin classes expressed by leukocytes, we report that β2 and α4 integrins use different RIAM-dependent and -independent pathways to undergo activation by talin.

Cluster of differentiation 43 deficiency in leukocytes leads to reduced atherosclerosis--brief report.

The aim of this study was to investigate the role of cluster of differentiation 43 (CD43), an integral membrane glycoprotein with both proadhesive and antiadhesive activities, in atherosclerosis. Low-density lipoprotein receptor-deficient mice were lethally irradiated and reconstituted with either bone marrow from CD43(-/-) mice or from wild-type controls. We found that mice lacking the CD43 on their leukocytes had significantly less severe atherosclerosis and that, contrary to our expectation, macrophage infiltration into the vessel wall was not affected by the lack of CD43 in the leukocytes. However, we found that CD43 mediates cholesterol homeostasis in macrophages by facilitating cholesterol efflux. This resulted in a significant reduction in storage of cholesterol in the aorta of mice lacking CD43 in the leukocytes. CD43 may be an important mediator of macrophage lipid homeostasis, thereby affecting macrophage foam cell formation and ultimately atherosclerotic plaque development.

Leukocyte Factor XII Mediates Inflammation and Its Deficiency Promotes Wound Healing

Abstract 616 Background:Older investigations suggest that Factor XII (FXII) influences the inflammatory response. FXII deficient patients have reduced leukocyte migration into skin windows. In addition to bradykinin generation, FXII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. In vitro, purified FXIIa corrects neutrophil aggregation and degranulation defects in FXII-deficient plasma. Our laboratory demonstrated that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation, growth and angiogenesis. Since FXII modulates wound angiogenesis, we examined the role for FXII in the inflammatory response and wound repair. Investigations:Exon 3 to 8–deleted FXII knockout mice (FXII−/−) were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited to the injury site per 20X high power field (HPF) was determined and analyzed by Image J (NIH). We observed that on Day 1, FXII−/− mice exhibit significantly decreased recruitment of CD11 b–labeled inflammatory cells to injury sites compared with wild type mice (WT) [p=0.0136]. Similar results are observed when uPAR KO mice were compared to WT [p=0.0001], suggesting that leukocyte migration in part is mediated through this receptor. Next we determined the nature of cells in the wound sites. FXII−/− mice have reduced wound neutrophils (Gr-1 staining) and macrophages (F4-80 staining). On the thioglycolate (TG)-induced peritoneal inflammation assay, the number of peritoneal exudate cells (PECs) was measured in WT and FXII−/− mice 1 and 7 days following instillation. FXII−/− mice exhibit significantly decreased number of PEC’s on days 1 and 7. Giemsa-Wright stain of peritoneal lavage fluid with manual differential counts or flow cytometry shows that there is a disproportionate decrease in neutrophil recruitment in peritoneal fluid of FXII−/− mice. Also, FXII−/− macrophages have reduced adherence to plastic. Identical assays performed on uPAR and bradykinin B2 receptor (B2R) KO mice did not reveal the same inflammatory defects; both uPAR and B2R KO mice have mild decreases in PEC cell numbers following TG instillation, but overall their inflammatory response remained intact. Investigations next determined if the leukocyte migration defect seen in FXII−/− mice is related to plasma FXII or an intrinsic leukocyte defect. On adoptive bone marrow (BM) transplantation experiments, WT bone marrow transplanted to FXII−/− mice corrects the TG-induced PEC migration defect after both 1 and 7 days from instillation. Alternatively, transplantation of FXII−/− bone marrow into WT mice produced a leukocyte migration defect. In order to further discriminate the contribution of FXII in leukocyte migration, we produced plasma FXII deficiency in WT mice using FXII siRNA. WT mice treated with FXII siRNA reduced plasma FXII activity to <10% at 24 h (T1/2= 6 h). These mice did not have a defect in PEC recruitment. In vivo infusion of purified FXII did not correct the leukocyte peritoneal migration defect in FXII−/− mice. Next we harvested BM from WT mice and isolated leukocytes to prepare bone marrow cell-derived mRNA. The mRNA was reverse-transcribed to cDNA and it demonstrates XII cDNA that shares sequence homology to hepatic XII from exons 2 through 14. Immunofluorescence staining of BM-derived leukocytes demonstrates XII-positive leukocytes with nuclear morphology resembling monocytes and neutrophils (Fig. 1). Finally although leukocyte FXII promotes their migration into wounds, FXII deficiency in leukocytes paradoxically is associated with improved rates of wound healing after punch biopsy. Conclusions:These data indicate that there is a unique pool of FXII in leukocytes distinct from plasma (hepatic) FXII. Leukocyte FXII, not plasma FXII, is responsible for leukocyte function in wounds and inflammation sites. FXII−/− mice have attenuated wound injury and, paradoxically, improved wound healing rates. Modulation of leukocyte FXII is a target for promotion of wound healing. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Collective action of hematopoietic cell subsets mediates anti-IL10R1 and CpG tumor immunity.

Based on the specificity of antigen recognition and the ability to generate long-lived memory responses, cancer immunotherapies primarily target tumor-associated T cells. Systemic administration of anti-IL-10R1 antibody in combination with local CpG administration has been shown to induce tumor regression in a T-cell-dependent manner. Here, we confirmed the anti-tumor efficacy of anti-IL-10R1 and CpG therapy in the highly aggressive B16F10 melanoma model. However, T cells were not required for tumor growth inhibition. Through cellular depletions and genetic models of leukocyte deficiency, we demonstrated that T, B, and NK cells, and neutrophils are not essential for anti-tumor efficacy. Nevertheless, hematopoietic cells as a whole are required for anti-IL-10R1- and CpG-induced tumor growth inhibition, suggesting that the collective action of multiple subsets of hematopoietic-derived cells is required for anti-tumor efficacy.

Deficiency of receptor-associated protein attenuates angiotensin II-induced atherosclerosis in hypercholesterolemic mice without influencing abdominal aortic aneurysms

Objective Receptor-associated protein (RAP) was initially described as a regulator of low density lipoprotein receptor-related protein 1 (LRP1), but is now known to regulate many proteins. Since the direct effects of RAP on vascular pathologies have not been studied, this study determined whether RAP deficiency influenced angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Methods and results Male LDL receptor −/− mice that were either RAP +/+ or −/− were infused with AngII (500 ng/kg/min) for 4 weeks while consuming a saturated fat-enriched diet. RAP deficiency had no effects on body weight or AngII-induced increases of systolic blood pressure. Despite increased plasma cholesterol concentrations, RAP deficiency reduced atherosclerotic lesion size in aortic arches, while having no effect on AngII-induced AAAs. RAP deficiency profoundly reduced LRP1 protein abundance in macrophages, but did not change its abundance in aortic smooth muscle cells. Also, RAP deficiency had no effects on mRNA abundance of LRP1 or lipoprotein lipase in macrophages. To determine whether RAP deficiency in leukocytes influenced AngII-induced atherosclerosis, irradiated male LDL receptor −/− mice were repopulated with bone marrow-derived cells from either RAP +/+ or −/− male mice. The chimeric mice were infused with AngII (500 ng/kg/min) for 4 weeks while fed the saturated fat-enriched diet. RAP deficiency in bone marrow-derived cells did not influence either plasma cholesterol concentrations or atherosclerotic lesion size. Conclusions Whole body RAP deficiency attenuated atherosclerosis without influencing AAAs in hypercholesterolemic mice infused with AngII. The anti-atherogenic effect was not attributable to RAP deficiency in bone marrow-derived cells.

MyD88 Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Independent of Signaling Through Toll-Like Receptors 2 and 4

The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis. AngII was infused into either apoE(-/-) or LDL receptor (LDLR)(-/-) male mice that were either MyD88(+/+) or (-/-). MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR(-/-) mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR(-/-) mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 strains. MyD88 deficiency in bone marrow-derived cells profoundly reduced both AngII-induced AAAs and atherosclerosis. However, TLR4 deficiency in bone marrow-derived cells had no effect on either pathology. These studies demonstrate that MyD88 deficiency in leukocytes profoundly reduces AngII-induced AAAs and atherosclerosis via mechanisms independent of either TLR2 or TLR4.

Wound healing defect of Vav3−/− mice due to impaired β2-integrin–dependent macrophage phagocytosis of apoptotic neutrophils

AbstractVav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for β2-integrins–mediated macrophage functions during wound healing. Vav3−/− and Vav1−/−/Vav3−/− mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3−/− bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with β2-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3−/−or β2-integrin(CD18)−/− macrophages into wound margins failed to restore the healing defect of Vav3−/− mice, suggesting Vav3 to control the β2-integrin–dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3−/− macrophages was causal for their reduced release of active transforming growth factor (TGF)-β1, for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-β1 deficiency in Vav3−/− macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-β1 into wounds of Vav3−/− mice fully rescued the delayed wound healing.

Role of Bone Marrow–Derived CC-Chemokine Receptor 5 in the Development of Atherosclerosis of Low-Density Lipoprotein Receptor Knockout Mice

CC chemokine receptor CCR5 is expressed by atheroma-associated cells and could mediate leukocyte attraction into developing lesions. We examined the role of bone marrow-derived CCR5 in the development of atherosclerotic lesions after 8, 12, or 35 weeks of high-fat diet. Low-density lipoprotein-receptor (LDLr)-deficient mice were lethally irradiated and transplanted with CCR5+/+ or CCR5-/- bone marrow. After 8 weeks of fat diet, CCR5 deficiency in leukocytes led to 30% decrease of macrophage accumulation within the fatty streak (P<0.05), with no change in lesion size. After 12 weeks of fat diet, CCR5 deficiency also resulted in 30% decrease of plaque-macrophage accumulation (P<0.005), associated with 16% reduction in lesion size in the aortic sinus (P=0.13), despite a significant increase in total cholesterol levels (P=0.03). Lesions with CCR5 deficiency showed 52% reduction in matrix metalloproteinase (MMP)-9 expression (P=0.02) and 2-fold increase in collagen accumulation (P<0.0001). These changes were associated with a significant increase of interleukin (IL)-10 mRNA expression in spleens of CCR5-/- mice compared with CCR5+/+ controls. In addition, we found enhanced IL-10 production by CCR5-deficient peritoneal macrophages and decreased tumor necrosis factor (TNF)-alpha production by CCR5-/- T cells in comparison with CCR5+/+ controls. CCR5-/- and CCR5+/+ reconstituted animals showed no differences in plaque size or composition after 35 weeks of high-fat diet despite the persistent absence of CCR5 in plaques of mice reconstituted with CCR5-/- bone marrow. Bone marrow-derived CCR5 favors the development of an inflammatory and collagen-poor plaque phenotype in association with decreased macrophage-derived IL-10 and enhanced T cell-derived TNF-alpha. These effects are not sustained in the very advanced stages of atherosclerosis.

Leukocyte-derived interleukin 10 is required for protection against atherosclerosis in low-density lipoprotein receptor knockout mice.

Atherosclerosis is an immunoinflammatory disease. Here we examined the role of leukocyte-derived interleukin 10 (IL-10) on advanced atherosclerosis development in low-density lipoprotein receptor knockout (LDLr-/-) mice. Bone marrow cells harvested from C57BL/6 IL-10-/- and IL-10+/+ mice were transplanted into irradiated male LDLr-/- mice. Four weeks after transplantation, mice were fed a high-fat cholate-free diet for 14 weeks. Despite no differences in weights, serum total, and HDL-cholesterol levels between the 2 groups, IL-10 deficiency in leukocytes induced a >2-fold increase in lesion development in the thoracic aorta compared with controls. We also found a significant 35% increase in aortic root lesion area of IL-10-/- mice compared with IL-10+/+ mice. Furthermore, IL-10 deficiency led to a marked increase in lymphocyte and macrophage accumulation associated with a significant reduction in collagen accumulation. Finally, transfer of IL-10-/- splenocytes to LDLr-/- mice resulted in a 3-fold increase in lesion size in the aortic sinus compared with mice transplanted with IL-10+/+ splenocytes. IL-10 expressed by leukocytes prevents exaggerated advanced atherosclerosis development and plays a critical role in modulation of cellular and collagen plaque composition, at least in part, through a modulation of the systemic immune response.