Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1(ADA)) or clade C (HIV-1(Indie-C1)) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1(Indie-C1)-infected macrophages recruited monocytes poorly in vitro compared with HIV-1(ADA)-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.

The aim of this study was to discover whether MCP-1/CCL2, a CC chemokine able to attract macrophages, is expressed in human pancreatic cancer and how it modulates cancer progression. All primary tumors were tested, and 6 of 14 pancreatic cancer cell lines were constitutively secreted CCL2. Analysis of the regulation demonstrated that the expression of CCL2 was significantly elevated and in a synergistic manner by IFN-gamma, tumor necrosis factor alpha, and interleukin 1beta. By immunohistochemistry and in situ hybridization, CCL2 production was confirmed in neoplastic ducts from surgical specimens. Serum levels of CCL2 in pancreatic cancer patients were significantly higher than in normal healthy subjects (P < 0.0001). Patients with high circulating levels of CCL2 had significantly higher survival rate than low CCL2 producers. Serum CCL2 levels positively correlated with tumor macrophage infiltration and inversely correlated with tumor proliferative activity (Ki67 expression). A direct effect of CCL2 on tumor cells is to be excluded, either because primary tumors as well as cell lines have no detectable CCL2 receptor (CCR2) and because addition of CCL2 on tumor cells in vitro did not modify cell cycle progression or apoptosis. In vitro, a model of tumor microenvironment showed a direct antiproliferative and proapoptotic activity of monocytes toward pancreatic cancer cell, which is mediated at least in part by interleukin 1beta. Moreover, other proinflammatory cytokines such as tumor necrosis factor alpha and IFN-gamma appeared able to induce apoptosis and to reduce the proliferative rate of pancreatic cancer. On the whole, the results presented in our investigation suggest that CCL2 could be a relevant negative regulator of pancreatic cancer progression.

Chemokines promote tumour progression by enhancing proliferation and modifying the immune response. The purpose of this study was to test the hypothesis that CCL2 monocyte chemotactic protein-1 (MCP-1) contributes to the progression of colorectal cancer by influencing the number and distribution of tumour associated macrophages (TAMs). Chemokine expression was assessed in human colorectal adenocarcinomas by ribonuclease protection assay (RPA). Colonic adenocarcinoma cell lines were used to assess chemokine production by enzyme linked immunosorbant assay (ELISA), and Boyden microchemotaxis assays were performed to determine cell line supernatant monocyte chemotactic activity. CCL2 production was assessed in paraffin embedded tumour samples by immunohistochemistry. Finally, the number of macrophages and their distribution was determined in the same colorectal adenocarcinomas and compared with CCL2 expression and tumour stage. Results showed that CCL2 produced by cell lines induced monocyte chemoattraction, the expression of this chemokine in solid cancers increased with tumour stage (P < 0.05) and immunohistochemistry localized production to tumour cells. Analysis of the macrophage infiltrate showed that the accumulation was significantly greater in tumours than controls (P < 0.005) and within tumours it was greatest in necrotic regions (median 44,600 per mm(3)). Macrophage accumulation increased with tumour stage and correlated with CCL2 expression (r(s) = 0.8). CXCL8 interleukin 8 (IL-8), a potent angiogenic factor and growth factor, was expressed in all tumours and cell lines. It is concluded that CCL2 induces the accumulation of tumour promoting TAMs in human colorectal cancer and represents a therapeutic target to modify the macrophage response and direct immune mediated therapy.

BACKGROUND

Heightened immune activation among human immunodeficiency virus (HIV)-infected persons may contribute to atherosclerosis. We assessed associations of serologic markers of monocyte activation, soluble CD163 (sCD163) and soluble CD14 (sCD14), and monocyte chemoattractant protein 1 (CCL2) with subclinical atherosclerosis among men with and those without HIV infection in the Multicenter AIDS Cohort Study.

METHODS

We performed noncontrast computed tomography on 906 men (566 HIV-infected men and 340 HIV-uninfected men), 709 of whom also underwent coronary computed tomographic angiography. Associations between each biomarker and the prevalence of coronary plaque, the prevalence of stenosis of ≥50%, and the extent of plaque were assessed by logistic and linear regression, adjusting for age, race, HIV serostatus, and cardiovascular risk factors.

RESULTS

Levels of all biomarkers were higher among HIV-infected men, of whom 81% had undetectable HIV RNA, and were associated with lower CD4(+) T-cell counts. In the entire population and among HIV-infected men, higher biomarker levels were associated with a greater prevalence of coronary artery stenosis of ≥50%. Higher sCD163 levels were also associated with greater prevalences of coronary artery calcium, mixed plaque, and calcified plaque; higher CCL2 levels were associated with a greater extent of noncalcified plaque.

CONCLUSIONS

sCD163, sCD14, and CCL2 levels were elevated in treated HIV-infected men and associated with atherosclerosis. Monocyte activation may increase the risk for cardiovascular disease in individuals with HIV infection.

Angiotensin II is the principle effector molecule of the renin angiotensin system (RAS). It exerts its various actions on the cardiovascular and renal system, mainly via interaction with the angiotensin II type-1 receptor (AT1R), which contributes to blood pressure regulation and development of hypertension but may also mediate effects on the immune system. Here we study the role of the RAS in myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), a model mimicking many aspects of multiple sclerosis. Quantitative RT-PCR analyses showed an up-regulation of renin, angiotensin-converting enzyme, as well as AT1R in the inflamed spinal cord and the immune system, including antigen presenting cells (APC). Treatment with the renin inhibitor aliskiren, the angiotensin II converting-enzyme inhibitor enalapril, as well as preventive or therapeutic application of the AT1R antagonist losartan, resulted in a significantly ameliorated course of MOG-EAE. Blockade of AT1R did not directly impact on T-cell responses, but significantly reduced numbers of CD11b+ or CD11c+ APC in immune organs and in the inflamed spinal cord. Additionally, AT1R blockade impaired the expression of CCL2, CCL3, and CXCL10, and reduced CCL2-induced APC migration. Our findings suggest a pivotal role of the RAS in autoimmune inflammation of the central nervous system and identify RAS blockade as a potential new target for multiple sclerosis therapy.

During inflammatory skin disorders such as psoriasis, atopic dermatitis, and allergic contact dermatitis, epidermal keratinocytes overexpress large amounts of soluble epidermal growth factor receptor ligands in response to tumor necrosis factor alpha and interferon gamma. These cytokines also promote de novo synthesis of numerous chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, in turn responsible for the recruitment of different leukocyte populations. This study demonstrates that stimulation of EGFR down-regulates CCL2, CCL5, and CXCL10, while it increases CXCL8 expression in keratinocytes. Conversely, EGFR signaling blockade produces opposite effects, with increased CCL2, CCL5, and CXCL10, and reduced CXCL8 expression. In a mouse model of contact hypersensitivity, a single topical administration of a selective EGFR kinase blocker before antigen challenge results in a markedly enhanced immune response with increased chemokine expression and heavier inflammatory cell infiltrate. Targeting EGFR on epithelial cells may thus have profound impact on inflammatory and immune responses.

Vaccine-based autoimmune (anti-amyloid) treatments are currently being examined for their therapeutic potential in Alzheimer's disease. In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 (triggering receptor expressed on myeloid cells 2) and the intracellular tolerance-associated transcript, Tmem176b (transmembrane domain protein 176b). In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation. Tmem176b expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. Transfection studies revealed that expression of TREM2 correlated negatively with motility, but correlated positively with the ability of microglia to stimulate CD4(+) T-cell proliferation, TNF (tumour necrosis factor) and CCL2 (chemokine ligand 2) production, but not IFNgamma (interferon gamma) production. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells. However, activating cells with LPS (lipopolysaccharide), but not IFNgamma, reduced the correlation between TREM2 expression and phagocytosis. Transfection of Tmem176b into both microglial and macrophage cell lines increased apoptosis. Taken together, these data suggest that, in vivo, Tmem176b(+) cells in closest apposition to amyloid may be the least able to clear amyloid. Conversely, the phagocytic TREM2(+) microglia on the plaque outer zones are positioned to capture and present self-antigens to CNS (central nervous system)-infiltrating lymphocytes without promoting pro-inflammatory lymphocyte responses. Instead, plaque-associated TREM2(+) microglia have the potential to evoke neuroprotective immune responses that may serve to support CNS function during pro-inflammatory anti-amyloid immune therapies.

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.

BACKGROUND

Although the statement that age is the greatest risk factor for Age-related macular degeneration (AMD) is widely accepted, the cellular and molecular explanations for that clinical statement are not generally known. A major focus of AMD research is the retinal pigment epithelium (RPE)/choroid. The purpose of this study was to characterize the changes in the RPE/choroid with age that may provide a background for the development of AMD.

RESULTS

We compared the transcriptional profiles, key protein levels and histology of the RPE/choroid from young and old mice. Using three statistical methods, microarray data demonstrated marked changes in the old mouse. There were 315 genes differentially expressed with age; most of these genes were related to immune responses and inflammatory activity. Canonical pathways having significant numbers of upregulated genes in aged RPE/choroid included leukocyte extravasation, complement cascades, natural killer cell signaling and IL-10 signaling. By contrast, the adjacent neural retina showed completely different age-related changes. The levels of proteins that participate in leukocyte extravasation and complement pathways were consistently increased in the normal, aged RPE/choroid. Furthermore, there was increased gene expression and protein levels of leukocyte attracting signal, chemokine ligand 2 (Ccl2) in aged RPE/choroid. In old animals, there was marked extravasation and accumulation of leukocytes from the choroidal circulation onto Bruch's membrane and into the RPE.

CONCLUSIONS

These phenotypic changes indicate that the RPE/choroid in the normal, old mouse has become an immunologically active tissue. There are signals from the normal, aged RPE/choroid which recruit leukocytes from the circulation and activate the complement cascade. These age-related changes that occur in the RPE/choroid with age, to the extent that they occur in the human retina, may provide the background for an error in regulation of immunological activity to cause AMD to appear in an elderly individual.

Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as <em>CCL2</em>, CCL3, CCL4, CCL5, and <em>CCL2</em>0, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 in the Kingdom of Saudi Arabia and has caused over 2400 cases and more than 800 deaths. Epidemiological studies identified diabetes as the primary comorbidity associated with severe and/or lethal MERS-CoV infection. Understanding how diabetes affects MERS is important due to the global burden of diabetes and pandemic potential of MERS-CoV. We used a model in which mice were made susceptible to MERS-CoV by expressing human DPP4 and type 2 diabetes was induced by administering a high fat diet. Upon infection with MERS-CoV, diabetic mice had a prolonged phase of severe disease and delayed recovery which was independent of virus titers. Histological analysis revealed that diabetic mice had delayed inflammation which was then prolonged through 21 dpi. Diabetic mice had fewer inflammatory monocyte/macrophages and CD4+ T cells which correlated with lower levels of Ccl2 and Cxcl10 expression. Diabetic mice also had lower levels of Tnfa, Il6, Il12b, and Arg1 expression and higher levels of Il17a expression. These data suggest that the increased disease severity observed in individuals with MERS and comorbid type 2 diabetes is likely due to a dysregulated immune response which results in more severe and prolonged lung pathology.

Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.

OBJECTIVE

To define the mechanisms underlying the accumulation of monocytes/macrophages in the islets of Langerhans.

METHODS

We tested the hypothesis that macrophage accumulation into the islets is caused by overexpression of the chemokine CCL2. To test this hypothesis, we generated transgenic mice and evaluated the cellular composition of the islets by immunohistochemistry and flow cytometry. We determined serum levels of CCL2 by enzyme-linked immunosorbent assay, determined numbers of circulating monocytes, and tested whether CCL2 could mobilize monocytes from the bone marrow directly. We examined development of diabetes over time and tested whether CCL2 effects could be eliminated by deletion of its receptor, CCR2.

RESULTS

Expression of CCL2 by beta-cells was associated with increased numbers of monocytes in circulation and accumulation of macrophages in the islets of transgenic mice. These changes were promoted by combined actions of CCL2 at the level of the bone marrow and the islets and were not seen in animals in which the CCL2 receptor (CCR2) was inactivated. Mice expressing higher levels of CCL2 in the islets developed diabetes spontaneously. The development of diabetes was correlated with the accumulation of large numbers of monocytes in the islets and did not depend on T- and B-cells. Diabetes could also be induced in normoglycemic mice expressing low levels of CCL2 by increasing the number of circulating myeloid cells.

CONCLUSIONS

These results indicate that CCL2 promotes monocyte recruitment by acting both locally and remotely and that expression of CCL2 by insulin-producing cells can lead to insulitis and islet destruction.

The expression of C1q, a recognition molecule of the complement system, is up-regulated following neuronal injury and is detected early in neurodegenerative disorders such as Alzheimer's disease. This multimeric protein triggers an enhancement of phagocytosis of suboptimally opsonized targets by microglia, the phagocytic cells of the CNS, similar to other phagocytes, enhances the uptake of apoptotic cells in peripheral phagocytes, and suppresses inflammatory cytokine production in human monocytes, macrophages and dendritic cells in the absence of activation of the entire complement cascade. The goal of this study was to determine if C1q could influence the inflammatory response to injury in the CNS, using primary rat microglia and neurons. The data show that microglia preferentially ingest apoptotic cells in comparison to live cells, like other professional phagocytes, that microglial ingestion of apoptotic neurons and neuronal blebs is enhanced by the presence of normal serum and that these enhanced levels of uptake are diminished in serum depleted of C1q. In addition, purified C1q bound to apoptotic neurons and neuronal blebs in a dose dependent manner, and alone triggered a significant enhancement of uptake by microglia. Microglia added to C1q coated wells or fed apoptotic neurons or neuronal blebs coated with C1q suppressed the lipopolysaccharide-induced production of proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and TNF-alpha, while the presence of C1q enhanced levels of the chemokine MCP-1/CCL2. The data are consistent with a protective role for C1q in the CNS during early stages of cell death by enhancing microglial clearance of apoptotic cells and suppressing proinflammatory cytokines.

OBJECTIVE

Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The pathways affected by tofacitinib and the effects on gene expression in situ are unknown. Therefore, tofacitinib effects on synovial pathobiology were investigated.

METHODS

A randomised, double-blind, phase II serial synovial biopsy study (A3921073; NCT00976599) in patients with RA with an inadequate methotrexate response. Patients on background methotrexate received tofacitinib 10 mg twice daily or placebo for 28 days. Synovial biopsies were performed on Days -7 and 28 and analysed by immunoassay or quantitative PCR. Clinical response was determined by disease activity score and European League Against Rheumatism (EULAR) response on Day 28 in A3921073, and at Month 3 in a long-term extension study (A3921024; NCT00413699).

RESULTS

Tofacitinib exposure led to EULAR moderate to good responses (11/14 patients), while placebo was ineffective (1/14 patients) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-3 (p<0.05) and chemokines CCL2, CXCL10 and CXCL13 (p<0.05). No overall changes were observed in synovial inflammation score or the presence of T cells, B cells or macrophages. Changes in synovial phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 strongly correlated with 4-month clinical responses (p<0.002). Tofacitinib significantly decreased plasma CXCL10 (p<0.005) at Day 28 compared with placebo.

CONCLUSIONS

Tofacitinib reduces metalloproteinase and interferon-regulated gene expression in rheumatoid synovium, and clinical improvement correlates with reductions in STAT1 and STAT3 phosphorylation. JAK1-mediated interferon and interleukin-6 signalling likely play a key role in the synovial response.

BACKGROUND

NCT00976599.

The monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 are key modulators of immune functions. In the nervous system, MCP-1/CCL2 is implicated in neuroinflammatory pathologies. However, cerebral functions of MCP-1/CCL2 under normal conditions are still unclear. In this study, using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific rat MCP-1 enzyme-linked immunosorbent assay (ELISA) approaches, we observed that MCP-1/CCL2 mRNA and protein were expressed in different punched regions of the normal rat central nervous system. Immunohistochemical studies further revealed that this chemokine is constitutively expressed not only in astrocytes but also in neurons, in discrete neuroanatomical regions. Neuronal expression of MCP-1/CCL2 is mainly found in the cerebral cortex, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, facial nuclei, motor and spinal trigeminal nuclei, and gigantocellular reticular nucleus and in Purkinje cells in the cerebellum. Moreover, we obtained the first evidence that MCP-1/CCL2 is constitutively expressed in cholinergic neurons, notably in the magnocellular preoptic and oculomotor nuclei, and in dopaminergic neurons of the substantia nigra pars compacta. In addition, in the lateral hypothalamic area, MCP-1/CCL2 co-localized with melanin-concentrating hormone-expressing neurons. Interestingly, we demonstrate a co-localization of MCP-1/CCL2 with vasopressin in magnocellular neuronal cell bodies and processes in the supraoptic and paraventricular hypothalamic nuclei, as well as in processes in the internal layer of the median eminence and in the posterior pituitary. Taken together, our data suggest that MCP-1/CCL2 could act as a modulator of neuronal activity and neuroendocrine functions.

Chemokines and their receptors are well described in the immune system, where they promote cell migration and activation. In the central nervous system, chemokine has been implicated in neuroinflammatory processes. However, an increasing number of evidence suggests that they have regulatory functions in the normal nervous system, where they could participate in cell communication. In this work, using a semiquantitative immunohistochemistry approach, we provide the first neuroanatomical mapping of constitutive neuronal CCR2 localization. Neuronal expression of CCR2 was observed in the anterior olfactory nucleus, cerebral cortex, hippocampal formation, caudate putamen, globus pallidus, supraoptic and paraventricular hypothalamic nuclei, amygdala, substantia nigra, ventral tegmental area, and in the brainstem and cerebellum. These data are largely in accordance with results obtained using quantitative autoradiography with [(125)I]MCP-1/CCL2 and RT-PCR CCR2 mRNA analysis. Furthermore, using dual fluorescent immunohistochemistry we studied the chemical phenotype of labeled neurons and demonstrated the coexistence of CCR2 with classical neurotransmitters. Indeed, localization of CCR2 immunostaining is observed in dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area as well as in cholinergic neurons in the substantia innominata and caudate putamen. Finally, we show that the preferential CCR2 ligand, MCP-1/CCL2, elicits Ca(2+) transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon. In conclusion, the constitutive neuronal CCR2 expression in selective brain structures suggests that this receptor could be involved in neuronal communication and possibly associated with cholinergic and dopaminergic neurotransmission and related disorders.

Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNbeta, IFNbeta-induced gene MxA mRNA, and TNFalpha. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPbeta, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPbeta on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.

Neuropathic pain after peripheral nerve injury, associated with local neuroinflammation in the spinal cord, is a severe incapacitating condition with which clinical treatment remains challenging. Inflammatory molecules signal through various intracellular transduction pathways, activation of which may amplify and cause spreading of the inflammatory response. We showed recently that spinal nerve lesion leads to rapid activation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signal transduction pathway in dorsal spinal cord microglia in relation with enhanced levels of spinal interleukin-6 (IL-6) protein. Here, we selectively inactivated JAK/STAT3 signaling in rat dorsal spinal cord glia through local, lentiviral-mediated production of the suppressor of cytokine signaling SOCS3, a physiologic inhibitory protein of JAK/STAT3, and analyzed its consequences in a preclinical model of neuropathic pain. The targeted blockade of JAK/STAT3 activity prevented the abnormal expression of IL-6, CC chemokine ligand CCL2, and activating transcription factor ATF3 induced in the spinal cord by chronic constriction injury of the sciatic nerve (CCI) and substantially attenuated mechanical hypersensitivity (allodynia) in rats. In naive rats, intrathecal administration of a proalgesic cytokine IL-6 rapidly activated microglial JAK/STAT3 and induced downstream changes closely resembling CCI-evoked alterations. We identified downstream mechanisms through which JAK/STAT3 pathway activation leads to the spreading of neuroinflammation. Our findings reveal that JAK/STAT3 signaling plays a major role in spinal cord plasticity and mechanical allodynia associated with peripheral nerve injury.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of <em>CCL2</em>, <em>CCL2</em>0, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.

OBJECTIVE

Glucagon-like peptide-1 (GLP-1), a member of the proglucagon-derived peptide family, was seen to exert favourable actions on cardiovascular function in preclinical and clinical studies. The mechanisms through which GLP-1 modulates cardiovascular function are complex and incompletely understood. We thus investigated whether the GLP-1 analogue, liraglutide, which is an acylated GLP-1, has protective effects on vascular endothelial cells.

METHODS

Nitrite and nitrate were measured in medium with an automated nitric oxide detector. Endothelial nitric oxide synthase (eNOS) activation was assessed by evaluating the phosphorylation status of the enzyme and evaluating eNOS activity by citrulline synthesis. Nuclear factor kappaB (NF-kappaB) activation was assessed by reporter gene assay.

RESULTS

Liraglutide dose-dependently increased nitric oxide production in HUVECs. It also caused eNOS phosphorylation, potentiated eNOS activity and restored the cytokine-induced downregulation of eNOS (also known as NOS3) mRNA levels, which is dependent on NF-kappaB activation. We therefore examined the effect of liraglutide on TNFalpha-induced NF-kappaB activation and NF-kappaB-dependent expression of proinflammatory genes. Liraglutide dose-dependently inhibited NF-kappaB activation and TNFalpha-induced IkappaB degradation. It also reduced TNFalpha-induced MCP-1 (also known as CCL2), VCAM1, ICAM1 and E-selectin mRNA expression. Liraglutide-induced enhancement of nitric oxide production and suppression of NF-kappaB activation were attenuated by the AMP-activated protein kinase (AMPK) inhibitor compound C or AMPK (also known as PRKAA1) small interfering RNA. Indeed, liraglutide induced phosphorylation of AMPK, which occurs through a signalling pathway independent of cyclic AMP.

CONCLUSIONS

Liraglutide exerts an anti-inflammatory effect on vascular endothelial cells by increasing nitric oxide production and suppressing NF-kappaB activation, partly at least through AMPK activation. These effects may explain some of the observed vasoprotective properties of liraglutide, as well as its beneficial effects on the cardiovascular system.

OBJECTIVE

To examine the effect of different sources of good manufacturing practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes.

METHODS

AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription-polymerase chain reaction or multiplex bead-based immunoassay. The role of different immunomodulators was analyzed.

RESULTS

All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2 ) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action.

CONCLUSIONS

The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Intracerebral hemorrhage (ICH) is a devastating type of stroke that lacks a specific treatment. An intense immune response develops after ICH, which contributes to neuronal injury, disability, and death. However, the specific mediators of inflammation-induced injury remain unclear. The objective of the present study was to determine whether blood-derived CCR2+ Ly6C(hi) inflammatory monocytes contribute to disability. ICH was induced in mice and the resulting inflammatory response was quantified using flow cytometry, confocal microscopy, and neurobehavioral testing. Importantly, blood-derived monocytes were distinguished from resident microglia by differential CD45 staining and by using bone marrow chimeras with fluorescent leukocytes. After ICH, blood-derived CCR2+ Ly6C(hi) inflammatory monocytes trafficked into the brain, outnumbered other leukocytes, and produced tumor necrosis factor. Ccr2(-/-) mice, which have few circulating inflammatory monocytes, exhibited better motor function following ICH than control mice. Chimeric mice with wild-type CNS cells and Ccr2(-/-) hematopoietic cells also exhibited early improvement in motor function, as did wild-type mice after inflammatory monocyte depletion. These findings suggest that blood-derived inflammatory monocytes contribute to acute neurological disability. To determine the translational relevance of our experimental findings, we examined CCL2, the principle ligand for the CCR2 receptor, in ICH patients. Serum samples from 85 patients were collected prospectively at two hospitals. In patients, higher CCL2 levels at 24 h were independently associated with poor functional outcome at day 7 after adjusting for potential confounding variables. Together, these findings suggest that inflammatory monocytes worsen early disability after murine ICH and may represent a therapeutic target for patients.