Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Ccl3 enhances docetaxel chemosensitivity in breast cancer by triggering proinflammatory macrophage polarization.

BACKGROUND: Although the antitumor efficacy of docetaxel (DTX) has long been attributed to the antimitotic activities, its impact on the tumor microenvironment (TME) has recently gained more attention. Macrophages are a major component of the TME and play a critical role in DTX efficacy; however, the underlying action mechanisms remain unclear. METHODS: DTX chemotherapeutic efficacy was demonstrated via both macrophage depletion and C-C motif chemokine ligand 3 (Ccl3)-knockout transgenic allograft mouse model. Ccl3-knockdown and Ccl3-overexpressing breast cancer cell allografts were used for the in vivo study. Combination therapy was used to evaluate the effect of Ccl3 induction on DTX chemosensitivity. Vital regulatory molecules and pathways were identified using RNA sequencing. Macrophage phagocytosis of cancer cells and its influence on cancer cell proliferation under DTX treatment were assessed using an in vitro coculture assay. Serum and tumor samples from patients with breast cancer were used to demonstrate the clinical relevance of our study. RESULTS: Our study revealed that Ccl3 induced by DTX in macrophages and cancer cells was indispensable for the chemotherapeutic efficacy of DTX. DTX-induced Ccl3 promoted proinflammatory macrophage polarization and subsequently facilitated phagocytosis of breast cancer cells and cancer stem cells. Ccl3 overexpression in cancer cells promoted proinflammatory macrophage polarization to suppress tumor progression and increase DTX chemosensitivity. Mechanistically, DTX induced Ccl3 by relieving the inhibition of cAMP-response element binding protein on Ccl3 via reactive oxygen species accumulation, and Ccl3 then promoted proinflammatory macrophage polarization via activation of the Ccl3-C-C motif chemokine receptor 5-p38/interferon regulatory factor 5 pathway. High CCL3 expression predicted better prognosis, and high CCL3 induction revealed better DTX chemosensitivity in patients with breast cancer. Furthermore, both the Creb inhibitor and recombinant mouse Ccl3 significantly enhanced DTX chemosensitivity. CONCLUSIONS: Our results indicate that Ccl3 induced by DTX triggers proinflammatory macrophage polarization and subsequently facilitates phagocytosis of cancer cells. Ccl3 induction in combination with DTX may provide a promising therapeutic rationale for increasing DTX chemosensitivity in breast cancer.

Development and Evaluation of an Ebola Virus Glycoprotein Mucin-Like Domain Replacement System as a New Dendritic Cell-Targeting Vaccine Approach against HIV-1.

The development of efficient vaccine approaches against HIV infection remains challenging in the vaccine field. Here, we developed an Ebola virus envelope glycoprotein (EboGP)-based chimeric fusion protein system and demonstrated that replacement of the mucin-like domain (MLD) of EboGP with HIV C2-V3-C3 (134 amino acids [aa]) or C2-V3-C3-V4-C4-V5-C5 (243 aa) polypeptides (EbGPΔM-V3 and EbGPΔM-V3-V5, respectively) still maintained the efficiency of EboGP-mediated viral entry into human macrophages and dendritic cells (DCs). Animal studies using mice revealed that immunization with virus-like particles (VLPs) containing the above chimeric proteins, especially EbGPΔM-V3, induced significantly more potent anti-HIV antibodies than HIV gp120 alone in mouse serum and vaginal fluid. Moreover, the splenocytes isolated from mice immunized with VLPs containing EbGPΔM-V3 produced significantly higher levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-4, IL-5, and macrophage inflammatory protein 1α (MIP-1α). Additionally, we demonstrated that coexpression of EbGPΔM-V3 and the HIV Env glycoprotein in a recombinant vesicular stomatitis virus (rVSV) vector elicited robust anti-HIV antibodies that may have specifically recognized epitopes outside or inside the C2-V3-C3 region of HIV-1 gp120 and cross-reacted with the gp120 from different HIV strains. Thus, this study has demonstrated the great potential of this DC-targeting vaccine platform as a new vaccine approach for improving immunogen delivery and increasing vaccine efficacy. IMPORTANCE Currently, there are more than 38.5 million reported cases of HIV globally. To date, there is no approved vaccine for HIV-1 infection. Thus, the development of an effective vaccine against HIV infection remains a global priority. This study revealed the efficacy of a novel dendritic cell (DC)-targeting vaccination approach against HIV-1. The results clearly show that the immunization of mice with virus-like particles (VLPs) and VSVs containing HIV Env and a fusion protein composed of a DC-targeting domain of Ebola virus GP with HIV C2-V3-C3 polypeptides (EbGPΔM-V3) could induce robust immune responses against HIV-1 Env and/or Gag in serum and vaginal mucosa. These findings provide a proof of concept of this novel and efficient DC-targeting vaccine approach in delivering various antigenic polypeptides of HIV-1 and/or other emergent infections to the host antigen-presenting cells to prevent HIV and other viral infections.

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.

Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform.

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

Fonsecaeapedrosoi Conidia and Hyphae Activate Neutrophils Distinctly: Requirement of TLR-2 and TLR-4 in Neutrophil Effector Functions.

Chromoblastomycosis is a chronic and progressive subcutaneous mycosis caused mainly by the fungus Fonsecaea pedrosoi. The infection is characterized by erythematous papules and histological sections demonstrating an external layer of fibrous tissue and an internal layer of thick granulomatous inflammatory tissue containing mainly macrophages and neutrophils. Several groups are studying the roles of the innate and adaptive immune systems in F. pedrosoi infection; however, few studies have focused on the role of neutrophils in this infection. In the current study, we verify the importance of murine neutrophils in the killing of F. pedrosoi conidia and hyphae. We demonstrate that phagocytosis and reactive oxygen species during infection with conidia are TLR-2- and TLR-4-dependent and are essential for conidial killing. Meanwhile, hyphal killing occurs by NET formation in a TLR-2-, TLR-4-, and ROS-independent manner. In vivo experiments show that TLR-2 and TLR-4 are also important in chromoblastomycosis infection. TLR-2KO and TLR-4KO animals had lower levels of CCL3 and CXCL1 chemokines and impaired neutrophil migration to the infected site. These animals also had higher fungal loads during infection with F. pedrosoi conidia, confirming that TLR-2 and TLR-4 are essential receptors for F. pedrosoi recognition and immune system activation. Therefore, this study demonstrates for the first time that neutrophil activation during F. pedrosoi is conidial or hyphal-specific with TLR-2 and TLR-4 being essential during conidial infection but unnecessary for hyphal killing by neutrophils.

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.

Correlation Analysis of Morphological Changes of Structural and Functional Areas of Mesenteric Lymph Nodes with Cytokines of Lymph of Thoracic Lymphatic Duct in Experimental Breast Cancer.

We performed a correlation analysis of the morphometric parameters of mesenteric lymph nodes and cytokine content in the lymph of thoracic duct in rats with chemically induced breast cancer. The study showed that activity of the local immune response in the lymph nodes in breast cancer is aimed at antitumor protection. In breast cancer, the area of the paracortical zone remained at the level of the intact group, while the area of lymphoid nodules with germinative centers and the area of medullary substance increased; the number of macrophages in the thymus-dependent zone and zone responsible for humoral immunity also increased. The following positive correlations were revealed: in germinative centers and medullary substance, number of mitotic cells correlated with cytokine IL-5 content and the number of medium lymphocytes correlated with the content of chemokine MIP-1α; in the germinative centers, the number of immunoblasts correlated with the level of cytokine GRO/KC, in the paracortical zone, the number of macrophages correlated with the level of chemokine MCP-1, the number of reticular cells correlated with IL-6 and M-CSF content; in medullary substance, the number of small lymphocytes and mature cells plasma cells (their content was reduced) correlated with the level of chemokine GRO/KC, which can be caused by their migration from the lymph node.

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.

CCL3 Signaling in the Tumor Microenvironment.

Within the tumor microenvironment, chemokines play a key role in immune cell trafficking regulation and immune landscape formulation. CCL3 or macrophage inflammatory protein-1α (MIP-1α), an important chemokine implicated in both immune surveillance and tolerance, has emerged as a prognostic biomarker in both solid and hematological malignancies. CCL3 exerts both antitumor and pro-tumor behavior which is context dependent highlighting the complexity of the underlying interrelated signaling cascades. Current CCL3-directed therapeutic approaches are investigational and further optimization is required to increase efficacy and minimize adverse events.

Blocking migration of regulatory T cells to leukemic hematopoietic microenvironment delays disease progression in mouse leukemia model.

Blocking the migration of regulatory T cells (Tregs) to the tumor microenvironment is a promising strategy for tumor immunotherapy. Treg accumulation in the leukemic hematopoietic microenvironment (LHME) has adverse impacts on patient outcomes. The mechanism and effective methods of disrupting Treg accumulation in the LHME have not been well established. Here, we studied the distribution and characteristics of Tregs in the LHME, investigated the effects of Treg ablation on leukemia progression, explored the mechanisms leading to Treg accumulation, and studied whether blocking Treg migration to the LHME delayed leukemia progression in MLL-AF9-induced mouse acute myeloid leukemia (AML) models using wildtype (WT) and Foxp3DTR/GFP mice. Increased accumulation of more activated Tregs was detected in the LHME. Inducible Treg ablation prolonged the survival of AML mice by promoting the antileukemic effects of CD8+ T cells. Furthermore, both local expansion and migration accounted for Treg accumulation in the LHME. Moreover, blocking the CCL3-CCR1/CCR5 and CXCL12-CXCR4 axes inhibited Treg accumulation in the LHME and delayed leukemia progression. Our findings provide laboratory evidence for a potential leukemia immunotherapy by blocking the migration of Tregs.

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.

Incorporation of Ebola glycoprotein into HIV particles facilitates dendritic cell and macrophage targeting and enhances HIV-specific immune responses.

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

Rhinovirus-induces progression of lung disease in a mouse model of COPD via IL-33/ST2 signaling axis.

Rhinovirus (RV), which is associated with acute exacerbations, also causes persistent lung inflammation in patients with chronic obstructive pulmonary disease (COPD), but the underlying mechanisms are not well-known. Recently, we demonstrated that RV causes persistent lung inflammation with accumulation of a subset of macrophages (CD11b+/CD11c+), and CD8+ T cells, and progression of emphysema. In the present study, we examined the mechanisms underlying the RV-induced persistent inflammation and progression of emphysema in mice with COPD phenotype. Our results demonstrate that at 14 days post-RV infection, in addition to sustained increase in CCL3, CXCL-10 and IFN-γ expression as previously observed, levels of interleukin-33 (IL-33), a ligand for ST2 receptor, and matrix metalloproteinase (MMP)12 are also elevated in mice with COPD phenotype, but not in normal mice. Further, MMP12 was primarily expressed in CD11b+/CD11c+ macrophages. Neutralization of ST2, reduced the expression of CXCL-10 and IFN-γ and attenuated accumulation of CD11b+/CD11c+ macrophages, neutrophils and CD8+ T cells in COPD mice. Neutralization of IFN-γ, or ST2 attenuated MMP12 expression and prevented progression of emphysema in these mice. Taken together, our results indicate that RV may stimulate expression of CXCL-10 and IFN-γ via activation of ST2/IL-33 signaling axis, which in turn promote accumulation of CD11b+/CD11c+ macrophages and CD8+ T cells. Furthermore, RV-induced IFN-γ stimulates MMP12 expression particularly in CD11b+/CD11c+ macrophages, which may degrade alveolar walls thus leading to progression of emphysema in these mice. In conclusion, our data suggest an important role for ST2/IL-33 signaling axis in RV-induced pathological changes in COPD mice.

Antibiotic Perturbation of Gut Microbiota Dysregulates Osteoimmune Cross Talk in Postpubertal Skeletal Development.

Commensal gut microbiota-host immune responses are experimentally delineated via gnotobiotic animal models or alternatively by antibiotic perturbation of gut microbiota. Osteoimmunology investigations in germ-free mice, revealing that gut microbiota immunomodulatory actions critically regulate physiologic skeletal development, highlight that antibiotic perturbation of gut microbiota may dysregulate normal osteoimmunological processes. We investigated the impact of antibiotic disruption of gut microbiota on osteoimmune response effects in postpubertal skeletal development. Sex-matched C57BL/6T mice were administered broad-spectrum antibiotics or vehicle-control from the age of 6 to 12 weeks. Antibiotic alterations in gut bacterial composition and skeletal morphology were sex dependent. Antibiotics did not influence osteoblastogenesis or endochondral bone formation, but notably enhanced osteoclastogenesis. Unchanged Tnf or Ccl3 expression in marrow and elevated tumor necrosis factor-α and chemokine (C-C motif) ligand 3 in serum indicated that the pro-osteoclastic effects of the antibiotics are driven by increased systemic inflammation. Antibiotic-induced broad changes in adaptive and innate immune cells in mesenteric lymph nodes and spleen demonstrated that the perturbation of gut microbiota drives a state of dysbiotic hyperimmune response at secondary lymphoid tissues draining local gut and systemic circulation. Antibiotics up-regulated the myeloid-derived suppressor cells, immature myeloid progenitor cells known for immunosuppressive properties in pathophysiologic inflammatory conditions. Myeloid-derived suppressor cell-mediated immunosuppression can be antigen specific. Therefore, antibiotic-induced broad suppression of major histocompatibility complex class II antigen presentation genes in bone marrow discerns that antibiotic perturbation of gut microbiota dysregulates critical osteoimmune cross talk.

Human Cervical Epithelial Cells Release Antiviral Factors and Inhibit HIV Replication in Macrophages.

The female reproductive tract is a major site of HIV sexual transmission. We here examined whether human cervical epithelial cells (HCEs) can be immunologically activated and produce antiviral factors against HIV. We demonstrated that HCEs (End1/E6E7 cells) possess the functional toll-like receptor (TLR)3 signaling system, which could be activated by Poly I:C and induce multiple cellular HIV restriction factors. The treatment of primary human macrophages with supernatant (SN) from TLR3-activated End1/E6E7 cell cultures resulted in HIV inhibition. This SN-mediated HIV inhibition was mainly through the induction of interferons (IFN)-ß and IFN-λs, as the antibodies to IFN-ß or IFN-λs receptor could effectively block the SN-mediated anti-HIV effect. Further studies showed that the incubation of macrophages with SN from the activated cervical epithelial cell cultures induced the expression of a number of IFN-stimulated genes (ISGs), including IFN-stimulated gene (ISG15), ISG56, 2', 5'-oligoadenylate synthetase 1 (OAS 1), OAS 2, Myxovirus Resistance A (MxA), MxB, and Guanylate-binding protein 5 (GBP5). In addition, TLR3-activated cells produced the CC chemokines [regulated on activation, normal T cell expressed and secreted (RANTES), Human macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1ß] the ligands of HIV entry co-receptor CCR5. These observations support further studies on HCEs as potentially crucial and alternative targets for immunological intervention to control and prevent HIV sexual transmission.

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte-M2 macrophage pathway.

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3+  CD8+ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1ß and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3+  CD8+ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1ß and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.

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.

Structural analysis and immunostimulatory potency of lipoteichoic acids isolated from three Streptococcus suis serotype 2 strains.

Streptococcus suis serotype 2 is an important porcine and human pathogen. Lipoteichoic acid (LTA) from S. suis has been suggested to contribute to its virulence, and absence of d-alanylation from the S. suis LTA is associated with increased susceptibility to cationic antimicrobial peptides. Here, using high-resolution NMR spectroscopy and MS analyses, we characterized the LTA structures from three S. suis serotype 2 strains differing in virulence, sequence type (ST), and geographical origin. Our analyses revealed that these strains possess-in addition to the typical type I LTA present in other streptococci-a second, mixed-type series of LTA molecules of high complexity. We observed a ST-specific difference in the incorporation of glycosyl residues into these mixed-type LTAs. We found that strains P1/7 (ST1, high virulence) and SC84 (ST7, very high virulence) can attach a 1,2-linked α-d-Glcp residue as branching substituent to an α-d-Glcp that is 1,3-linked to glycerol phosphate moieties and that is not present in strain 89-1591 (ST25, intermediate virulence). In contrast, the latter strain could glycosylate its LTA at the glycerol O-2 position, which was not observed in the other two strains. Using LTA preparations from WT strains and from mutants with an inactivated prolipoprotein diacylglyceryl transferase, resulting in deficient lipoprotein acylation, we show that S. suis LTAs alone do not induce Toll-like receptor 2-dependent pro-inflammatory mediator production from dendritic cells. In summary, our study reveals an unexpected complexity of LTAs present in three S. suis serotype 2 strains differing in genetic background and virulence.