Bone is the most frequent site of breast cancer metastasis, and once such metastasis occurs, complete remission is extremely difficult to achieve. In an effort to define the mechanisms underlying metastatic spread of breast cancer to bone, we previously developed and characterized the highly bone metastatic 4T1E/M3 mouse breast cancer cells. We found that following injection into mice, 4T1E/M3 cells exhibited greater bone metastasis and greater in vitro anchorage-independent growth and cell migration than their parental cells (4T1E). We also found that expression of intracellular adhesion molecule-1 (ICAM-1) is crucially involved in these metastatic activities of 4T1E/M3 cells. In the present study, our analysis of gene and protein expression revealed that production of chemokine CCL2 (MCP-1) is dramatically reduced in 4T1E/M3 cells, and that restoration of CCL2 expression in 4T1E/M3 cells diminishes their metastasis to bone and lung. Overexpression of CCL2 in 4T1E/M3 cells significantly reduced not only in vitro anchorage-independent cell growth and cell migration, but also mRNA and cell surface expression of ICAM-1. Conversely, knocking down CCL2 in 4T1E parental cells augmented their metastatic spread to spine and lung. The expression of ICAM-1 was also upregulated in 4T1E-derived CCL2 knockdown cells. Taken together, these results suggest that CCL2 expression may negatively regulate breast cancer metastasis to bone marrow and lung in our model and that expression of ICAM-1 plays a crucial role in that process.

Disseminated leishmaniasis (DL) is an emerging form of Leishmania braziliensis infection characterised by multiple cutaneous lesions on different parts of the body and a high rate of mucosal involvement. Systemic production of TNFα and IFNγ in DL patients is lower than in cutaneous leishmaniasis (CL) caused by L. braziliensis, which may account for parasite dissemination due to the decreased ability to control parasite growth. In this study, the systemic and in situ immune response of DL and CL patients was characterised through evaluation of chemokine and cytokine production. In situ evaluation showed similar production of IFNγ, TNFα, IL-10, transforming growth factor-beta (TGFβ), chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL11 and chemokine (C-X-C motif) ligand 10 (CXCL10) in papular and ulcerative lesions from DL as well as in ulcerated lesions from CL. Serum levels of CXCL9, a chemokine that attracts T-cells, was higher in serum from DL than from CL. These data indicate that a decrease in the type 1 immune response in peripheral blood of DL patients is due to attraction of Leishmania antigen-activated T-cells to the multiple cutaneous lesions. This may account for the absence of or few parasites in the lesions and for the development of ulcers similar to those observed in CL.

Chromosome 17 is known to contain TB susceptibility genes. Polymorphisms in two of these genes, namely NOS2A and CCL2, have been associated with TB in various populations. To investigate a possible association of gene variants with TB in the South African Coloured population we genotyped SNPs from NOS2A and CCL2 in over 800 TB cases and controls. We found a significant association between TB and two haplotypes, containing the functional rs9282799 and rs8078340 SNPs, in the NOS2A promoter. The T allele of rs8078340, found in the haplotype over-represented in cases (p=0.015, p(c)=0.038, OR=1.4, 95% CI [1.1-1.8]), was previously shown to decrease the quantity of DNA-protein complex bound as well as the duration of binding and may decrease nitric oxide (NO) production. The C allele of rs8078340 was present in the haplotype more frequent in controls (p=0.011, p(c)=0.029, OR=1.4, 95% CI [1.1-1.8]). In the single-point analysis of NOS2A, rs2779249 (previously associated with TB in Brazilians) and the functional rs8078340 were nominally associated with disease. No association was found between any of the other SNPs or haplotypes studied and TB. This study presents evidence that haplotypes in the NOS2A promoter influence susceptibility to TB and confirms the importance of NO production in the disease.

Obesity-associated chronic inflammation is characterized by an accumulation of adipose tissue macrophages (ATMs). It is generally believed that those macrophages are derived from peripheral blood monocytes. However, recent studies suggest that local proliferation of macrophages is responsible for ATM accumulation. In the present study, we revealed that both migration and proliferation contribute to ATM accumulation during obesity development. We show that there is a significant increase in ATMs at the early stage of obesity, which is largely due to an enhanced in situ macrophage proliferation. This result was obtained by employing fat-shielded irradiation and bone marrow reconstitution. Additionally, the production of CCL2, a pivotal chemoattractant of monocytes, was not found to be increased at this stage, corroborating with a critical role of proliferation. Nonetheless, as obesity proceeds, the role of monocyte migration into adipose tissue becomes more significant and those new immigrants further proliferate locally. These proliferating ATMs mainly reside in crown-like structures formed by macrophages surrounding dead adipocytes. We further showed that IL-4/STAT6 is a driving force for ATM proliferation. Therefore, we demonstrated that local proliferation of resident macrophages contributes to ATM accumulation during obesity development and has a key role in obesity-associated inflammation.

Early-life seizures result in increased susceptibility to seizures and greater neurologic injury with a second insult in adulthood. The mechanisms which link seizures in early-life to increased susceptibility to neurologic injury following a 'second hit' are not known. We examined the contribution of microglial activation and increased proinflammatory cytokine production to the subsequent increase in susceptibility to neurologic injury using a kainic acid (KA)-induced, established 'two-hit' seizure model in rats. Postnatal day (P)15 rats were administered intraperitoneal KA (early-life seizures) or saline, followed on P45 with either a 'second hit' of KA, a first exposure to KA (adult seizures), or saline. We measured the levels of proinflammatory cytokines (IL-1 beta, TNF-alpha, and S100B), the chemokine CCL2, microglial activation, seizure susceptibility and neuronal outcomes in adult rats 12 h and 10 days after the second hit on P45. The 'two-hit' group exposed to KA on both P15 and P45 had higher levels of cytokines, greater microglial activation, and increased susceptibility to seizures and neurologic injury compared to the adult seizures group. Treatment after early-life seizures with Minozac, a small molecule experimental therapeutic that targets upregulated proinflammatory cytokine production, attenuated the enhanced microglial and cytokine responses, the increased susceptibility to seizures, and the greater neuronal injury in the 'two-hit' group. These results implicate microglial activation as one mechanism by which early-life seizures contribute to increased vulnerability to neurologic insults in adulthood, and indicate the potential longer term benefits of early-life intervention with therapies that target up-regulation of proinflammatory cytokines.

BACKGROUND

Inflammation caused by the Lyme disease spirochete B. burgdorferi is an important factor in the pathogenesis of Lyme neuroborreliosis. Our central hypothesis is that B. burgdorferi can cause disease via the induction of inflammatory mediators such as cytokines and chemokines in glial and neuronal cells. Earlier we demonstrated that interaction of B. burgdorferi with brain parenchyma induces inflammatory mediators in glial cells as well as glial (oligodendrocyte) and neuronal apoptosis using ex vivo and in vivo models of experimentation.

METHODS

In this study we evaluated the ability of live B. burgdorferi to elicit inflammation in vitro in differentiated human MO3.13 oligodendrocytes and in differentiated primary human oligodendrocytes, by measuring the concentration of immune mediators in culture supernatants using Multiplex ELISA assays. Concomitant apoptosis was quantified in these cultures by the in situ terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) assay and by quantifying active caspase-3 by flow cytometry. The above phenomena were also evaluated after 48 h of stimulation with B. burgdorferi in the presence and absence of various concentrations of the anti-inflammatory drug dexamethasone.

RESULTS

B. burgdorferi induced enhanced levels of the cytokine IL-6 and the chemokines IL-8 and CCL2 in MO3.13 cells as compared to basal levels, and IL-8 and CCL2 in primary human oligodendrocytes, in a dose-dependent manner. These cultures also showed significantly elevated levels of apoptosis when compared with medium controls. Dexamethasone reduced both the levels of immune mediators and apoptosis, also in a manner that was dose dependent.

CONCLUSIONS

This finding supports our hypothesis that the inflammatory response elicited by the Lyme disease spirochete in glial cells contributes to neural cell damage. As oligodendrocytes are vital for the functioning and survival of neurons, the inflammation and subsequent apoptosis of oligodendrocytes induced by B. burgdorferi could contribute to the pathogenesis of Lyme neuroborreliosis.

Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor-associated macrophages (TAMs) within the TME play a crucial role in endocrine resistance. Here, we aimed to elucidate the relationship between TAMs and the endocrine-resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen-sensitive (MCF7-S) or -resistant (MCF7-R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAMs, and then treated with tamoxifen. CCL2 immunohistochemistry was performed on pathological sections from 100 patients with invasive estrogen receptor-positive breast cancer. We found that macrophages cultured in the CM of MCF7-S and MCF7-R cells were induced into TAMs, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAMs secreted CCL2, which increased endocrine resistance in breast cancer cells via activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with the infiltration of CD163+ macrophages (r = 0.548, P < 0.001), and patients with high CCL2 expression presented shorter progression-free survival than those with low CCL2 expression (P < 0.05). We conclude that CCL2 secreted by TAMs activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAMs may be novel therapeutic targets for patients with endocrine-resistant breast cancer.

E-selectin is a surface marker of endothelial cell (EC) inflammation, one of the hallmarks of atherogenesis. Thus, we tested the hypothesis that delivery of microRNA (miR)-146a and miR-181b with an E-selectin-targeting multistage vector (ESTA-MSV) to inflamed endothelium covering atherosclerotic plaques inhibits atherosclerosis. Cy5-conjugated miR-146a and miR-181b were packaged in polyethylene glycol-polyethyleneimine (PEG/PEI) nanoparticles and loaded into ESTA-MSV microparticles. Both miRs were downregulated in tumor necrosis factor (TNF)-α-treated ECs. Transfection of TNF-α-treated mouse aortas and cultured ECs with miRs was more efficient with ESTA-MSV than with the PEG/PEI. Likewise, miR-146a/-181b packaged in ESTA-MSV efficiently suppressed the chemokines, CCL2, CCL5, CCL8, and CXCL9, and monocyte adhesion to ECs. Complementary in vivo tests were conducted in male apolipoprotein E-deficient mice fed a Western diet and injected intravenously with the particles prepared as above biweekly for 12 weeks. Treatment with miRs packaged in ESTA-MSV but not in PEG/PEI reduced atherosclerotic plaque size. Concurrently, vascular inflammation markers, including macrophages in aortic root lesions and chemokine expression in aortic tissues were reduced while the vascular smooth muscle cells and collagen increased in plaques from ESTA-MSV/miRs-treated vs. vehicle-treated mice. Our data supported our hypothesis that ESTA-MSV microparticle-mediated delivery of miR-146a/-181b ameliorates endothelial inflammation and atherosclerosis.

Rationale: Uncontrolled inflammatory innate response and impaired adaptive immune response are associated with clinical severity in patients with Coronavirus disease 2019 (COVID-19).

Objectives: To compare the immunopathology of COVID-19 acute respiratory distress syndrome (ARDS) to that of non-COVID-19 ARDS, and to identify biomarkers associated with mortality in patients with COVID-19 ARDS.

Methods: Prospective observational monocenter study. Immunocompetent patients diagnosed with RT-PCR-confirmed SARS-CoV-2 infection and ARDS admitted between March 8 and March 30, 2020 were included and compared with patients with non-COVID-19 ARDS. The primary clinical endpoint of the study was mortality at day 28. Flow cytometry analyses and serum cytokines measurements were performed at days 1-2 and 4-6 of ICU admission.

<strong class="sub-title"> Measurements and main results: </strong> As compared with patients with non-COVID-19 ARDS (n=36), those with COVID-19 (n=38) were not significantly different regarding age, gender, and SOFA and SAPS II scores but exhibited a higher day-28 mortality (34% vs 11%, p=0.030). COVID-19 patients showed profound and sustained T CD4+ (p=0.002), CD8+ (p<0.0001) and B (p<0.0001) lymphopenia, higher HLA-DR expression on monocytes (p<0.001) and higher serum concentrations of EGF, GM-CSF, IL-10, <em>CCL2</em>/MCP-1, CCL3/MIP-1a, CXCL10/IP-10, CCL5/RANTES, and <em>CCL2</em>0/MIP-3a. After adjusting on age and SOFA, serum CXCL10/IP-10 (p=0.047) and GM-CSF (p=0.050) were higher and naso-pharyngeal RT-PCR cycle threshold values lower (p=0.010) in COVID-19 patients who were dead at day-28.

Conclusions: Profound global lymphopenia and a "chemokine signature" were observed in COVID-19 ARDS. Increased serum concentrations of CXCL10/IP-10 and GM-CSF, together with higher naso-pharyngeal SARS-CoV-2 viral load, were associated with day-28 mortality. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: SARS-CoV-2, COVID-19, ARDS, chemokines, cytokines.

Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the β cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to β cells favoring apoptosis. Inactivation of these miRNAs in recipient β cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in β cells. The induction of these genes may promote the recruitment of immune cells and exacerbate β cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)(-/-) mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS(-/-) mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage.

CCR2, and its principle ligand MCP-1/CCL2, have been well documented for their ability to induce monocyte infiltration and promote the pathogenesis of rheumatoid arthritis and atherosclerosis. In order to assess additional roles for CCR2, we inserted allogeneic implants into CCR2-/- and MCP-1-/- mice and characterized T cell responses and the regulatory role of CCR2 on MCP-1 expression. The results demonstrate a marked decrease in lymphocyte infiltration in both CCR2-/- and MCP-1-/- animals. In contrast, IL-12 and CTL function were only suppressed in CCR2-/- animals. Further, whereas MCP-1 was only transiently elevated in the inflammatory fluid of WT animals, levels were sustained within the implants (5000pg/ml; >8 days) and serum (243pg/ml) of CCR2-/- mice. Higher levels of MCP-1 were also observed in the culture supernatants of CCR2-/- macrophages as compared to WT cells despite no difference in mRNA levels. Evidence that MCP-1 levels are regulated by receptor binding and internalization was suggested by its rapid decline when added to WT macrophages at 37 degrees C but not 4 degrees C. These studies indicate that CCR2 plays an important role in regulating T cell responses and controlling the level of MCP-1 at inflammatory sites.

BACKGROUND

During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite.

RESULTS

We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-alpha expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression.

CONCLUSIONS

These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.

Although intestinal epithelial cells are known to up-regulate the expression of several chemokine genes in response to the stimulation with B. fragilis enterotoxin (BFT), there has been little understanding on the cellular mechanisms of BFT-induced mucosal inflammation. To test whether nuclear transcriptional factor-kappa B (NF-kappaB) is involved in the process, we stimulated intestinal epithelial cells with BFT, and evaluated the signalling NF-kappaB pathways. BFT increased signals of NF-kappaB in HT-29 and T84 epithelial cell lines as well as primary human colon epithelial cells. NF-kappaB molecules activated by BFT stimulation were composed of p65 and p50 heterodimers. In contrast, BFT decreased the signals of IkappaBalpha and IkappaB epsilon, as assessed by immunoblot. Super-repressors of IkappaBalpha, IkappaB kinase (IKK)beta, and NF-kappaB inducing kinase (NIK) inhibited an up-regulated transcription of downstream target gene (CXCL8) of NF-kappaB. Moreover, blocking the activation of NF-kappaB by MG-132 or antisense p50 oligonucleotide transfection resulted in down-regulated expression of chemokines such as CXCL1, CXCL8, and CCL2 in BFT-stimulated HT-29 cells. In addition, NF-kappaB inhibition suppressed the BFT-induced neutrophil transepithelial migration in T84 cells. These results indicate that NF-kappaB can be a central regulator of chemokine gene expression in BFT-stimulated intestinal epithelial cells and may be an important regulator of neutrophil migration.

OBJECTIVE

Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA.

METHODS

The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists.

RESULTS

VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively.

CONCLUSIONS

VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.

Ozone exposure produces acute inflammation and neutrophil influx in the distal lung. Alveolar epithelial cells cover a large surface area, secrete chemokines, and may initiate or modify the inflammatory response. The effect of ozone on chemokine production by these cells has not been defined. Isolated rat type II cells were cultured in different conditions to express the morphologic appearance and biochemical markers for the type I and the type II cell phenotypes. These cells were exposed to ozone at an air/liquid interface. The type I-like cells were more susceptible to injury than the type II cells and showed signs of injury at exposure levels of 100 ppb ozone for 60 min. Both phenotypes showed evidence of lipid peroxidation after ozone exposure as measured by 8-isoprostane production, but neither phenotype secreted increased amounts of MIP-2 (CXCL3), CINC-1 (CXCL1), or MCP-1 (CCL2) in response to ozone. Both cell phenotypes secreted MIP-2 and MCP-1 in response to IL-1beta or lipopolysaccharide, but there was no priming or synergy with ozone. It is likely that the inflammatory response to ozone in the alveolar compartment is not due to the direct effect of ozone on epithelial cells.

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.

One of the defining features of the majority of FOXP3(+) Tregs is their inability to produce typical T-cell-derived cytokines. Little is known, however, about their capacity to produce chemokines. As Tregs are constitutively present in, and rapidly traffic to, non-lymphoid tissues, we hypothesized that they may produce chemokines to direct the composition of cells that infiltrate inflamed tissues. Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL-8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2-5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8-dependent mechanism. These data provide the first evidence that although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells.

OBJECTIVE

Chemokines are central to the innate immune response following tissue damage, injury and some diseases. The function of chemokines in nervous system autoimmune diseases has been long recognized. There is also growing evidence that disease-associated or injury-induced functional expression of chemokines/receptors in both neural and nonneural elements of the peripheral nervous system play crucial roles in the pathophysiology of chronic pain.

RESULTS

Chemokine involvement in neuropathic pain processing has recently been established in animal models. Evidence of chemokine contribution to chronic pain includes the upregulation of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its respective receptor, CCR2, in many subpopulations of sensory neurons. Activation of CCR2 by MCP-1 elicits membrane depolarization, triggers action potentials and sensitizes nociceptors via transactivation of transient receptor potential channels TRPA1 and TRPV1. Increased signaling by stromal-derived factor-1 (SDF-1/CXCL12) and its receptor, CXCR4, has been shown to contribute to chronic pain behavior. The use of specific chemokine receptor antagonists for CCR2 and CXCR4 successfully reverses nociceptive pain behavior.

CONCLUSIONS

Our results suggest that specific chemokines/receptors are upregulated by sensory neurons following peripheral nerve injury and appear to participate in neural signal processing leading to chronic pain states. Taken together, chemokines and their receptors are potential targets for development of novel therapeutics.

Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.

OBJECTIVE

We investigated the association of anti-inflammatory cytokine interleukin (IL)-1 receptor antagonist (IL-1ra), adiponectin, proinflammatory cytokines IL-1 beta, IL-6, and CCL2, and tumor necrosis factor-alpha with beta-cell function, metabolic status, and clinical remission in patients with recent-onset type 1 diabetes.

METHODS

Serum was obtained from 256 newly diagnosed patients (122 males and 134 females, median age 9.6 years). Stimulated C-peptide, blood glucose, and A1C were determined in addition to circulating concentration of cytokines at 1, 6, and 12 months after diagnosis. Analyses were adjusted for sex, age, and BMI percentile.

RESULTS

Anti-inflammatory IL-1ra was positively associated with C-peptide after 6 (P = 0.0009) and 12 (P = 0.009) months. The beneficial association of IL-1ra on beta-cell function was complemented by the negative association of IL-1 beta with C-peptide after 1 month (P = 0.009). In contrast, anti-inflammatory adiponectin was elevated in patients with poor metabolic control after 6 and 12 months (P < 0.05) and positively correlated with A1C after 1 month (P = 0.0004). Proinflammatory IL-6 was elevated in patients with good metabolic control after 1 month (P = 0.009) and showed a positive association with blood glucose disposal after 12 months (P = 0.047).

CONCLUSIONS

IL-1ra is associated with preserved beta-cell capacity in type 1 diabetes. This novel finding indicates that administration of IL-1ra, successfully improving beta-cell function in type 2 diabetes, may also be a new therapeutic approach in type 1 diabetes. The relation of adiponectin and IL-6 with remission and metabolic status transfers observations from in vitro and animal models into the human situation in vivo.

Proinflammatory circulating monocytes have important roles in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Yet there is limited information on their accumulation in blood during disease, the mechanisms that regulate their infiltration into the central nervous system (CNS), and whether medications affect their biology. We found a significant and prolonged elevation of CD11b(+)CCR2(+)Ly6C(high) proinflammatory monocytes in the blood of mice by the second day of immunization for EAE. At onset of clinical signs, levels of proinflammatory monocytes plummeted to those in naive mice. At day 16, when the majority of mice were at peak disease severity, clinical scores were inversely correlated to the proportion of proinflammatory monocytes in blood, and directly correlated with that in the spinal cord. Treatment with the MS medication laquinimod prevented EAE, correspondent with retention of proinflammatory monocytes in blood. The reduced entry of proinflammatory monocytes into the CNS by laquinimod was attributed to reduction of their levels of CD62L and matrix metalloproteinase-9. Moreover, the spinal cord of laquinimod-treated mice did not have elevated levels of CCR2 and CCL2, which provide chemotactic cues for monocytes. These results shed light on the important role of the trafficking of proinflammatory monocytes into the CNS to promote disease activity, and they identify a mechanism of action of laquinimod in MS.

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.