Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice.

AIMS: To explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. MAIN METHODS: The unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. KEY FINDINGS: IL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SIGNIFICANCE: CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.

A Novel Resolution of Diabetes: C-C Chemokine Motif Ligand 4 Is a Common Target in Different Types of Diabetes by Protecting Pancreatic Islet Cell and Modulating Inflammation.

Systemic inflammation is related to hyperglycemia in diabetes mellitus (DM). C-C chemokine motif ligand (CCL) 4 is upregulated in type 1 & type 2 DM patients. This study aimed to investigate if CCL4 could be a potential target to improve blood sugar control in different experimental DM models. Streptozotocin-induced diabetic mice, Leprdb /JNarl diabetic mice, and C57BL/6 mice fed a high fat diet were used as the type 1 DM, type 2 DM, and metabolic syndrome model individually. Mice were randomly assigned to receive an anti-CCL4 neutralizing monoclonal antibody. The pancreatic ß-cells were treated with streptozotocin for in vitro experiments. In streptozotocin-induced diabetic mice, inhibition of CCL4 controlled blood sugar, increased serum insulin levels, increased islet cell proliferation and decreased pancreatic interleukin (IL)-6 expression. In the type 2 diabetes and metabolic syndrome models, CCL4 inhibition retarded the progression of hyperglycemia, reduced serum tumor necrosis factor (TNF)-α and IL-6 levels, and improved insulin resistance via reducing the phosphorylation of insulin receptor substrate-1 in skeletal muscle and liver tissues. CCL4 inhibition directly protected pancreatic ß-cells from streptozotocin stimulation. Furthermore, CCL4-induced IL-6 and TNF-α expressions could be abolished by siRNA of CCR2/CCR5. In summary, direct inhibition of CCL4 protected pancreatic islet cells, improved insulin resistance and retarded the progression of hyperglycemia in different experimental models, suggesting the critical role of CCL4-related inflammation in the progression of DM. Future experiments may investigate if CCL4 could be a potential target for blood sugar control in clinical DM.

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation.

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2-expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4+ T cells in the lung. We discovered lung-resident CD4+ T cells that expressed a disease-specific public CDR3ß T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2-CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2-CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4.

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

Mycobacterium abscessus Clearance by Neutrophils Is Independent of Autophagy.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly prevalent in chronic lung disease, including cystic fibrosis, and infections are characterized by neutrophil-dominated environments. However, mechanisms of immune control are poorly understood. Azithromycin, a macrolide antibiotic with immunomodulatory effects, is used to treat M. abscessus infections. Recently, inhibition of macrophage bactericidal autophagy was described for azithromycin, which could be detrimental to the host. Therefore, we explored the role of autophagy in mycobactericidal neutrophils. Azithromycin did not affect M. abscessus-induced neutrophil reactive oxygen species formation, phagocytosis, or cytokine secretion, and neutrophils treated with azithromycin killed M. abscessus equally as well as untreated neutrophils from either healthy or cystic fibrosis subjects. One clinical isolate was killed more effectively in azithromycin-treated neutrophils, suggesting that pathogen-specific factors may interact with an azithromycin-sensitive pathway. Chloroquine and rapamycin, an inhibitor and an activator of autophagy, respectively, also failed to affect mycobactericidal activity, suggesting that autophagy was not involved. However, wortmannin, an inhibitor of intracellular trafficking, inhibited mycobactericidal activity, but as a result of inhibiting phagocytosis. The effects of these autophagy-modifying agents and azithromycin in neutrophils from healthy subjects were similar between the smooth and rough morphotypes of M. abscessus However, in cystic fibrosis neutrophils, wortmannin inhibited killing of a rough clinical isolate and not a smooth isolate, suggesting that unique host-pathogen interactions exist in cystic fibrosis. These studies increase our understanding of M. abscessus virulence and of neutrophil mycobactericidal mechanisms. Insight into the immune control of M. abscessus may provide novel targets of therapy.

CCL4 Signaling in the Tumor Microenvironment.

CCL4, a CC chemokine, previously known as macrophage inflammatory protein (MIP)-1ß, has diverse effects on various types of immune and nonimmune cells by the virtue of its interaction with its specific receptor, CCR5, in collaboration with related but distinct CC chemokines such as CCL3 and CCL5, which can also bind CCR5. Several lines of evidence indicate that CCL4 can promote tumor development and progression by recruiting regulatory T cells and pro-tumorigenic macrophages, and acting on other resident cells present in the tumor microenvironment, such as fibroblasts and endothelial cells, to facilitate their pro-tumorigenic capacities. These observations suggest the potential efficacy of CCR5 antagonists for cancer treatment. On the contrary, under some situations, CCL4 can enhance tumor immunity by recruiting cytolytic lymphocytes and macrophages with phagocytic ability. Thus, presently, the clinical application of CCR5 antagonists warrants more detailed analysis of the role of CCL4 and other CCR5-binding chemokines in the tumor microenvironment.

CCL3/CCR1 mediates CD14+CD16- circulating monocyte recruitment in knee osteoarthritis progression.

OBJECTIVES: Monocyte-derived macrophages, as the predominant immune cell type that is increased in inflamed synovium, play a vital role during knee osteoarthritis (KOA) progression. However, the mechanisms underlying the recruitment of circulating monocytes to osteoarthritic knees remain uncertain. Based on previous data obtained from plasma, we investigated the contributions of CCL2, CCL3, CCL4 and their cognate receptors in circulating monocyte chemotaxis and KOA development. METHODS: Using flow cytometry staining, we characterized the expression patterns of the chemokine receptors in CD14+CD16- circulating monocytes from KOA patients and healthy volunteers. The expression of chemokines in synovial fluids, synovium and cartilage was investigated in KOA patients and in patients without KOA. The role of chemokines and their cognate receptors in the chemotaxis of CD14+CD16- circulating monocytes was assessed using chemokine neutralizing antibodies (NA) and receptor antagonists in vitro and in vivo. RESULTS: The majority of CD14+CD16- circulating monocytes were CCR1-and CCR2-positive. CCL2, CCL3 and CCL4 were elevated in synovial fluid of KOA patients compared with that of controls. The most likely source of these chemokines is inflamed synovium and cartilage in the osteoarthritic knee. The CCL3/CCR1 and CCL2/CCR2 axes showed substantial ability to recruit CD14+CD16- monocytes in transwell assays. Similar results were confirmed in a mouse model of collagenase-induced KOA (CIA) in which blocking either the CCL3/CCR1 axis or the CCL2/CCR2 axis reduced synovial hyperplasia and F4/80+ macrophage infiltration. CONCLUSIONS: Our findings suggested that, analogous to the CCL2/CCR2 axis, CCL3 produced in osteoarthritic knees can chemoattract circulating monocytes to the inflamed synovium through CCR1.

A Four-Chemokine Signature Is Associated with a T-cell-Inflamed Phenotype in Primary and Metastatic Pancreatic Cancer.

PURPOSE: The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC. EXPERIMENTAL DESIGN: Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing. RESULTS: Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this "4-chemokine signature" positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell-inflamed phenotype across primary PDAC and PDAC liver metastases. CONCLUSIONS: A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

Recruitment of CD103+ dendritic cells via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy.

Although a clinical breakthrough for cancer treatment, it remains that a minority of patients respond to checkpoint inhibitor (CPI) immunotherapy. The composition of tumor-infiltrating immune cells has been identified as a key factor influencing CPI therapy success. Thus, enhancing tumor immune cell infiltration is a critical challenge. A lack of the chemokine CCL4 within the tumor microenvironment leads to the absence of CD103+ dendritic cells (DCs), a crucial cell population influencing CPI responsiveness. Here, we use a tumor stroma-targeting approach to deliver CCL4; by generating a fusion protein of CCL4 and the collagen-binding domain (CBD) of von Willebrand factor, we show that CBD fusion enhances CCL4 tumor localization. Intravenous CBD-CCL4 administration recruits CD103+ DCs and CD8+ T cells and improves the antitumor effect of CPI immunotherapy in multiple tumor models, including poor responders to CPI. Thus, CBD-CCL4 holds clinical translational potential by enhancing efficacy of CPI immunotherapy.

Exploring the immunomodulatory role of depot medroxyprogesterones acetate and endogenous progesterone levels in HIV infected and uninfected women.

OBJECTIVE: The aim of this proof of concept study was to determine the effect of depot medroxyprogesterone acetate on host and viral factors in HIV infected and uninfected women. RESULTS: In this study, the gene expression levels for CCL5, CCR5 and CXCR4 was significantly higher in HIV positive women when compared to HIV negative women (p < 0.05). An upregulation of CCR5 and CXCR4 was evident in less than 20% of the HIV infected women and none of the HIV uninfected women. The mean fold change for CCL3 was much higher in HIV uninfected when compared to infected women with a borderline significance (p = 0.062). In HIV uninfected women, the mean fold change in CCL3, CCL4, and CCL5 gene expression was not statistically different between women on DMPA versus women not on hormonal contraception. The proportion of women with an upregulation of CCL4 and CCR5 was higher in HIV infected women on DMPA. There was no association between endogenous progesterone level and chemokines and the HIV-1 receptors. The gene expression levels in the chemokine receptors CCR5 and CXCR4 were significantly higher in the HIV infected women when compared to the women who remained HIV uninfected.

The modulatory effects of the PDE4 inhibitors CHF6001 and roflumilast in alveolar macrophages and lung tissue from COPD patients.

BACKGROUND: We compared the anti-inflammatory effects of phosphodiesterase type 4 (PDE4) inhibitor roflumilast with CHF6001, a novel PDE4 inhibitor designed for inhaled administration, using human alveolar macrophages (AM) and lung tissue explants models. METHODS: AM from 13 chronic obstructive pulmonary disease (COPD) patients and 10 smoking controls and lung tissue from 7 COPD patients were stimulated with LPS following preincubation with roflumilast (0.000001-10 µM), CHF6001 (0.000001-0.1 µM), or vehicle. After 24 h, supernatants were analysed for cytokines by ELISA. The effects of both compounds on the phosphorylation and cellular localisation of cAMP response element binding protein (CREB) were assessed by immunofluorescence and Western blot analysis. Extracted RNA was used for quantitative PCR analysis of PDE4 A, B and D mRNA. RESULTS: PDE4 A, B and D expression were increased in alveolar macrophages and lung tissue of COPD patients compared to controls. Roflumilast and CHF6001 significantly reduced TNF-α production in AM and lung tissue. CHF6001 was more potent than roflumilast with lower EC50s of 0.02, 0.01 and 0.31 nM compared to 0.87, 0.47 and 10.8 nM in respective samples. PDE4 inhibition also inhibited secretion of the chemokines CCL2 and CCL4 from macrophages. Both compounds increased nuclear levels of phosphorylated CREB. CONCLUSION: PDE4 inhibitors caused a robust anti-inflammatory effect on TNF-α production from COPD AM, with inhibition of selective chemokines also observed. CHF6001 caused more potent inhibition of TNF-α production from COPD AM and lung tissue compared to roflumilast.

In Human Immunodeficiency Virus primary infection, early combined antiretroviral therapy reduced γδ T-cell activation but failed to restore their polyfunctionality.

Primary and chronic human immunodeficiency virus (HIV) infection alters γδ T-cell features. However, there is no evidence about early combined antiretroviral therapy (cART) and γδ T-cell dynamics. In the present study, HIV-positive individuals were divided into those with early primary infection (EPI) and those with late primary infection (LPI). The analysis of γδ T cells was performed by flow cytometry before and after therapy. Polyfunctional profile was assessed after in vitro peripheral blood mononuclear cell (PBMC) exposure to specific antigens. The results show that primary infection induced an expansion of Vδ1 T cells in LPI. Before treatment, a massive activation of γδ T-cell subsets was observed in both groups of patients, that correlated with disease progression and was significantly reduced after cART introduction. Despite this, CD107A-expressing Vδ1 T cells in both groups were significantly fewer than in healthy donors, but were restored by therapy introduction. Polyfunctional analysis of Vδ1 T cells from HIV-positive individuals revealed a lower frequency of CD107A+  CCL-4+ Vδ1 T-cell subsets than healthy donors that persists after therapy. Functional profile of Vδ2 was similar to that in healthy donors before therapy but, at 6 months, a lower frequency of CD107A, interferon-γ- or tumor necrosis factor-α-producing Vδ2 T cells was observed in the EPI group. Finally, individuals with LPI showed a lower frequency of quadruple-functional Vδ2 T-cell subset. In conclusion, during primary HIV infection, the baseline Vδ1 T-cell activation is correlated with immune reconstitution potential. Moreover, an altered γδ polyfunctional profile occurred, persisting after cART. Further studies are needed to understand whether a longer treatment of primary infection may increase γδ T-cell functionality.

Immune imbalance is associated with the development of preeclampsia.

Preeclampsia (PE) is characterized by hypertension and proteinuria. It affects about 5% to 8% of pregnancies and causes maternal and perinatal mortality and morbidity. The immune imbalance and excessive inflammatory response play vital roles in the pathogenesis of PE.In this study, we performed a case-control study to investigate the levels of cytokines, chemokines and adhesion molecules in serum and placenta of normal pregnant and PE women by Bio-Plex multiplex immunoassay and immunohistochemistry. In addition, we explored the phenotypes of monocyte and macrophage in peripheral blood and placentas in 2 groups by using flow cytometry analysis and immunohistochemistry.Our results show that pro-inflammatory factors, including interleukin-1ß (IL-1ß), IL-6, IL-7, IL-8, IL-17a, monocyte chemotactic protein 1 (MCP -1), and macrophage inflammatory protein 1ß (MIP-1ß) were significantly increased in serum of women with PE compared with controls. In addition, we detected that IL-1ß, IL-6, and MCP-1 were also increased in placentas of women with PE. We further revealed that peripheral blood monocytes showed a pro-inflammatory M1-like phenotype in women with PE. Consistently, M1 macrophage infiltration was increased in placenta of women with PE compared to that of normal pregnant women.Our results demonstrated that immune imbalance promotes an inflammatory state during PE and it may be a potential therapeutic possibility for the management of PE.

Critical role of CCL4 in eosinophil recruitment into the airway.

BACKGROUND: Excessive eosinophil airway infiltration is a clinically critical condition in some cases. Eosinophilic pneumonia (EP) is a pulmonary condition involving eosinophil infiltration of the lungs. Although several chemokines, including eotaxin-1 (CCL11), RANTES (CCL5) and macrophage inflammatory protein 1ß (MIP-1ß or CCL4), have been detected in bronchoalveolar lavage fluid (BALF) from patients with EP, the pathophysiological mechanisms underlying EP, including potential relationships between eosinophils and CCL4, have not been fully elucidated. OBJECTIVE: To examine the involvement of CCL4 in eosinophilic airway inflammation. METHODS: We analysed supernatants of activated eosinophils and BALF from 16 patients with eosinophilic pneumonia (EP). Further, we examined the effects of CCL4 on eosinophil functions in vitro and those of anti-CCL4 neutralizing antibody in an in vivo model. RESULTS: We found that purified human eosinophils stimulated with IL-5 predominantly secreted CCL4 and that patients with EP had elevated CCL11 and CCL4 levels in BALF compared with samples from individuals without EP. Because CCL4 levels were more strongly correlated with eosinophil count and expression of eosinophil granule proteins than CCL11, in vitro experiments using purified eosinophils concentrated on the former chemokine. Interestingly, CCL4 acted as a chemoattractant for eosinophils. In a mouse model, administration of a CCL4-neutralizing antibody attenuated eosinophilic airway infiltration and airway hyperresponsiveness. CONCLUSIONS AND CLINICAL RELEVANCE: Overall, these findings highlight an important role of CCL4 in the mechanisms underlying eosinophil recruitment into the airway and may provide a novel insight into this potential therapeutic target.

TroCCL4, a CC chemokine of Trachinotus ovatus, is involved in the antimicrobial immune response.

CC chemokines are a large subfamily of chemokines that play an important role in the innate immune system. To date, several CC chemokines have been identified in fish species; however, the activities and functions of these putative chemokines remain ambiguous in teleosts, especially in the golden pompano, Trachinotus ovatus. Here, we characterized CC chemokine ligand 4 from T. ovatus (TroCCL4) and studied its functions. TroCCL4 contains a 294 bp open reading frame that encodes a putative peptide comprising 97 amino acids. TroCCL4 shares a high amino acid sequence similarity of 31.11%-78.35% with other CC chemokines sequences in humans and teleosts and has four cysteine residues that are conserved among other CC chemokines. TroCCL4 is also related to the macrophage inflammatory protein (MIP) group of CC chemokines. TroCCL4 expression was most abundant in immune organs and significantly upregulated in a time-dependent manner following Edwardsiella tarda infection. Recombinant TroCCL4 (rTroCCL4) induced the migration of peripheral blood leukocytes and the cellular proliferation of head kidney lymphocytes. In addition, rTroCCL4 inhibited the growth of Escherichia coli and E. tarda, indicating an antimicrobial function. Furthermore, the results of in vivo analysis showed that TroCCL4 overexpression in T. ovatus significantly enhanced macrophage activation; upregulated the gene expression of interleukin 1-ß (IL-1ß), interleukin 15 (IL15), interferon-induced Mx protein (Mx), tumor necrosis factor α (TNFα), complement C3, and major histocompatibility complex (MHC) class Iα and class IIα; and protected against bacterial infection in fish tissues. In contrast, knockdown of TroCCL4 expression resulted in increased bacterial dissemination and colonization in fish tissues. Taken together, our results provide evidence indicating that TroCCL4 has the ability to stimulate leukocytes and macrophages and enhance host immunity to defend against bacterial infection.

Microglial translational profiling reveals a convergent APOE pathway from aging, amyloid, and tau.

Alzheimer's disease (AD) is an age-associated neurodegenerative disease characterized by amyloidosis, tauopathy, and activation of microglia, the brain resident innate immune cells. We show that a RiboTag translational profiling approach can bypass biases due to cellular enrichment/cell sorting. Using this approach in models of amyloidosis, tauopathy, and aging, we revealed a common set of alterations and identified a central APOE-driven network that converged on CCL3 and CCL4 across all conditions. Notably, aged females demonstrated a significant exacerbation of many of these shared transcripts in this APOE network, revealing a potential mechanism for increased AD susceptibility in females. This study has broad implications for microglial transcriptomic approaches and provides new insights into microglial pathways associated with different pathological aspects of aging and AD.

Leishmania donovani mediated higher expression of CCL4 induces differential accumulation of CD4+CD56+NKT and CD8+CD56+NKT cells at infection site.

Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP-1ß was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF-ß dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF-ß producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF-ß expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.

Baboon CD8 T cells suppress SIVmac infection in CD4 T cells through contact-dependent production of MIP-1α, MIP-1ß, and RANTES.

Simian immunodeficiency virus (SIV) infection in rhesus macaques is often characterized by high viremia and CD4 T cell depletion. By contrast, SIV infection in African nonhuman primate natural hosts is typically nonpathogenic despite active viral replication. Baboons are abundant in Africa and have a geographical distribution that overlaps with natural hosts, but they do not harbor SIVs. Previous work has demonstrated baboons are resistant to chronic SIV infection and/or disease in vivo but the underlying mechanisms remain unknown. Using in vitro SIVmac infections, we sought to identify SIV restriction factors in baboons by comparing observations to the pathogenic rhesus macaque model. SIVmac replicated in baboon PBMC but had delayed kinetics compared to rhesus PBMC. However, SIVmac replication in baboon and rhesus isolated CD4 cells were similar to the kinetics seen for rhesus PBMC, demonstrating intracellular restriction factors do not play a strong role in baboon inhibition of SIVmac replication. Here, we show CD8 T cells contribute to the innate SIV-suppressive activity seen in naïve baboon PBMC. As one mechanism of restriction, we identified higher production of MIP-1α, MIP-1ß, and RANTES by baboon PBMC. Contact between CD4 and CD8 T cells resulted in maximum production of these chemokines and suppression of viral replication, whereas neutralization of CCR5-binding chemokines in baboon PBMC increased viral loads. Our studies indicate baboon natural restriction of SIVmac replication is largely dependent on CD4-extrinsinc mechanisms mediated, in part, by CD8 T cells.