Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4-6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

OBJECTIVE

We hypothesized that the type 1 IFNs would play a pivotal role in antiglioma immunosurveillance through promotion of type 1 adaptive immunity and suppression of immunoregulatory cells.

METHODS

We induced de novo gliomas in Ifnar1(-/-) (deficient for type 1 IFN receptors) or wild-type mice by intracerebroventricuar transfection of NRas and a short hairpin RNA against P53 using the Sleeping Beauty transposon system. We analyzed the survival of 587 glioma patients for single nucleotide polymorphisms (SNP) in type 1 IFN-related genes.

RESULTS

Ifnar1(-/-) mice exhibited accelerated tumor growth and death. Analyses of brain tumor-infiltrating lymphocytes in Ifnar1(-/-) mice revealed an increase of cells positive for CD11b(+)Ly6G(+) and CD4(+)FoxP3(+), which represent myeloid-derived suppressor cells and regulatory T cells, respectively, but a decrease of CD8(+) cytotoxic T lymphocytes (CTLs) compared with wild-type mice. Ifnar1(-/-) mouse-derived glioma tissues exhibited a decrease in mRNA for the CTL-attracting chemokine Cxcl10, but an increase of <em>Ccl2</em> and <em>Ccl2</em>2, both of which are known to attract immunoregulatory cell populations. Dendritic cells generated from the bone marrow of Ifnar1(-/-) mice failed to function as effective antigen-presenting cells. Moreover, depletion of Ly6G(+) cells prolonged the survival of mice with developing gliomas. Human epidemiologic studies revealed that SNPs in IFNAR1 and IFNA8 are associated with significantly altered overall survival of patients with WHO grade 2 to 3 gliomas.

CONCLUSIONS

The novel Sleeping Beauty-induced murine glioma model led us to discover a pivotal role for the type 1 IFN pathway in antiglioma immunosurveillance and relevant human SNPs that may represent novel prognostic markers.

The transcription factor nuclear factor kappa B (NF-kappaB) is a key regulator of inflammatory processes in reactive glial cells. We utilized a transgenic mouse model (GFAP-IkappaBalpha-dn) where the classical NF-kappaB pathway is inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (IkappaBalpha) in glial fibrillary acidic protein (GFAP)-expressing cells, which include astrocytes, Schwann cells, and satellite cells of the dorsal root ganglion (DRG) and sought to determine whether glial NF-kappaB inhibition leads to a reduction in pain behavior and inflammation following chronic constriction injury (CCI) of the sciatic nerve. As expected, following CCI nuclear translocation, and hence activation, of NF-kappaB was detected only in the sciatic nerve of wild type (WT) mice, and not in GFAP-IkappaBalpha-dn mice, while upregulation of GFAP was observed in the sciatic nerve and DRGs of both WT and GFAP-IkappaBalpha-dn mice, indicative of glial activation. Following CCI, mechanical and thermal hyperalgesia were reduced in GFAP-IkappaBalpha-dn mice compared to those in WT, as well as gene and protein expression of CCL2, CCR2 and CXCL10 in the sciatic nerve. Additionally, gene expression of TNF, CCL2, and CCR2 was reduced in the DRGs of transgenic mice compared to those of WT after CCI. We can therefore conclude that transgenic inhibition of NF-kappaB in GFAP-expressing glial cells attenuated pain and inflammation after peripheral nerve injury. These findings suggest that targeting the inflammatory response in Schwann cells and satellite cells may be important in treating neuropathic pain.

Interleukin-1β (IL-1β) is abundant in the tumor microenvironment, where this cytokine can promote tumor growth, but also antitumor activities. We studied IL-1β during early tumor progression using a model of orthotopically introduced 4T1 breast cancer cells. Whereas there is tumor progression and spontaneous metastasis in wild-type (WT) mice, in IL-1β-deficient mice, tumors begin to grow but subsequently regress. This change is due to recruitment and differentiation of inflammatory monocytes in the tumor microenvironment. In WT mice, macrophages heavily infiltrate tumors, but in IL-1β-deficient mice, low levels of the chemokine CCL2 hamper recruitment of monocytes and, together with low levels of colony-stimulating factor-1 (CSF-1), inhibit their differentiation into macrophages. The low levels of macrophages in IL-1β-deficient mice result in a relatively high percentage of CD11b⁺ dendritic cells (DCs) in the tumors. In WT mice, IL-10 secretion from macrophages is dominant and induces immunosuppression and tumor progression; in contrast, in IL-1β-deficient mice, IL-12 secretion by CD11b⁺ DCs prevails and supports antitumor immunity. The antitumor immunity in IL-1β-deficient mice includes activated CD8⁺ lymphocytes expressing IFN-γ, TNF-α, and granzyme B; these cells infiltrate tumors and induce regression. WT mice with 4T1 tumors were treated with either anti-IL-1β or anti-PD-1 Abs, each of which resulted in partial growth inhibition. However, treating mice first with anti-IL-1β Abs followed by anti-PD-1 Abs completely abrogated tumor progression. These data define microenvironmental IL-1β as a master cytokine in tumor progression. In addition to reducing tumor progression, blocking IL-1β facilitates checkpoint inhibition.

After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.

The generalized seizures of status epilepticus (SE) trigger a series of molecular and cellular events that produce cognitive deficits and can culminate in the development of epilepsy. Known early events include opening of the blood-brain barrier (BBB) and astrocytosis accompanied by activation of brain microglia. Whereas circulating monocytes do not infiltrate the healthy CNS, monocytes can enter the brain in response to injury and contribute to the immune response. We examined the cellular components of innate immune inflammation in the days following SE by discriminating microglia vs. brain-infiltrating monocytes. Chemokine receptor 2 (CCR2(+)) monocytes invade the hippocampus between 1 and 3 d after SE. In contrast, only an occasional CD3(+) T lymphocyte was encountered 3 d after SE. The initial cellular sources of the chemokine CCL2, a ligand for CCR2, included perivascular macrophages and microglia. The induction of the proinflammatory cytokine IL-1β was greater in FACS-isolated microglia than in brain-invading monocytes. However, Ccr2 knockout mice displayed greatly reduced monocyte recruitment into brain and reduced levels of the proinflammatory cytokine IL-1β in hippocampus after SE, which was explained by higher expression of the cytokine in circulating and brain monocytes in wild-type mice. Importantly, preventing monocyte recruitment accelerated weight regain, reduced BBB degradation, and attenuated neuronal damage. Our findings identify brain-infiltrating monocytes as a myeloid-cell subclass that contributes to neuroinflammation and morbidity after SE. Inhibiting brain invasion of CCR2(+) monocytes could represent a viable method for alleviating the deleterious consequences of SE.

In mouse models of lung metastasis, before the appearance of significant metastases, localized changes in vascular permeability have been observed, which appear to set the stage for tumour growth. However, it is unclear whether this is also true in human patients. Here, we show that MD-2, a coreceptor for Toll-like receptor 4 that has a key role in the innate immune response, triggers the formation of regions of hyperpermeability in mice by upregulating C-C chemokine receptor type 2 (CCR2) expression. The CCR2-CCL2 system induces the abundant secretion of permeability factors such as serum amyloid A3 and S100A8. Disruption of MD-2 or CCR2 abrogates the formation of hyperpermeable regions, resulting in reduced tumour cell homing. Furthermore, fibrinogen, which is processed during permeability-mediated coagulation, is also localized in areas of elevated CCR2 expression in tumour-bearing human lungs. Our findings raise the possibility that CCR2 upregulation might represent a marker for regions of increased susceptibility to metastatic homing in lung cancer.

OBJECTIVE

The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines.

METHODS

Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting.

RESULTS

Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic.

CONCLUSIONS

Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1β). The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides.

OBJECTIVE

Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.

The intestinal epithelium is continuously renewed by intestinal epithelial stem cells (IESCs) positioned at the base of each crypt. Mesenchymal-derived factors are essential to maintain IESCs; however, the cellular composition and development of such mesenchymal niche remains unclear. Here, we identify pericryptal CD34+ Gp38+ αSMA- mesenchymal cells closely associated with Lgr5+ IESCs. We demonstrate that CD34+ Gp38+ cells are the major intestinal producers of the niche factors Wnt2b, Gremlin1, and R-spondin1, and are sufficient to promote maintenance of Lgr5+ IESCs in intestinal organoids, an effect mainly mediated by Gremlin1. CD34+ Gp38+ cells develop after birth in the intestinal submucosa and expand around the crypts during the third week of life in mice, independently of the microbiota. We further show that pericryptal CD34+gp38+ cells are rapidly activated by intestinal injury, up-regulating niche factors Gremlin1 and R-spondin1 as well as chemokines, proinflammatory cytokines, and growth factors with key roles in gut immunity and tissue repair, including IL-7, Ccl2, Ptgs2, and Amphiregulin. Our results indicate that CD34+ Gp38+ mesenchymal cells are programmed to develop in the intestine after birth to constitute a specialized microenvironment that maintains IESCs at homeostasis and contribute to intestinal inflammation and repair after injury.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new pandemic infectious disease that originated in China. COVID-19 is a global public health emergency of international concern. COVID-19 causes mild to severe illness with high morbidity and mortality, especially in preexisting risk groups. Therapeutic options are now limited to COVID-19. The hallmark of COVID-19 pathogenesis is the cytokine storm with elevated levels of interleukin-6 (IL-6), IL-1β, tumor necrosis factor-alpha (TNF-α), chemokine (C-C-motif) ligand 2 (CCL2), and granulocyte-macrophage colony-stimulating factor (GM-CSF). COVID-19 can cause severe pneumonia, and neurological disorders, including stroke, the damage to the neurovascular unit, blood-brain barrier disruption, high intracranial proinflammatory cytokines, and endothelial cell damage in the brain. Mast cells are innate immune cells and also implicated in adaptive immune response, systemic inflammatory diseases, neuroinflammatory diseases, traumatic brain injury and stroke, and stress disorders. SARS-CoV-2 can activate monocytes/macrophages, dendritic cells, T cells, mast cells, neutrophils, and induce cytokine storm in the lung. COVID-19 can activate mast cells, neurons, glial cells, and endothelial cells. SARS-CoV-2 infection can cause psychological stress and neuroinflammation. In conclusion, COVID-19 can induce mast cell activation, psychological stress, cytokine storm, and neuroinflammation.

Keywords: COVID-19; angiotensin-converting enzyme 2; cytokine storm; mast cells; neuroinflammation; severe acute respiratory syndrome coronavirus 2; traumatic brain injury.

OBJECTIVE

Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses.

METHODS

K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation.

RESULTS

K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases.

CONCLUSIONS

These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

BACKGROUND

Designing sustainable animal production systems that better balance productivity and resistance to disease is a major concern. In order to address questions related to immunity and resistance to disease in pig, it is necessary to increase knowledge on its immune system and to produce efficient tools dedicated to this species.

RESULTS

A long-oligonucleotide-based chip referred to as SLA-RI/NRSP8-13K was produced by combining a generic set with a newly designed SLA-RI set that targets all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex. The chip was used to study the immune response of pigs following stimulation of porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin for 24 hours. Transcriptome analysis revealed that ten times more genes were differentially expressed after PMA/ionomycin stimulation than after LPS stimulation. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than of B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) genes. In addition, a very intense repression of THBS1 (thrombospondin 1) was observed. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing. Repression of MHC class II genes was observed after both stimulations. Our results provide preliminary data suggesting that antisense transcripts mapping to the SLA complex may have a role during immune response.

CONCLUSIONS

The SLA-RI/NRSP8-13K chip was found to accurately decipher two distinct immune response activations of PBMCs indicating that it constitutes a valuable tool to further study immunity and resistance to disease in pig. The transcriptome analysis revealed specific and common features of the immune responses depending on the stimulation agent that increase knowledge on pig immunity.

OBJECTIVE

Interleukin-10 knockout (IL-10(-/-)) mice spontaneously develop colitis characterized by T-helper cell type 1-polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor alpha (PPARalpha) ligand fenofibrate, and the PPARdelta ligand GW0742, in IL-10(-/-) mice and investigated the cellular/molecular mechanisms for fenofibrate action.

METHODS

The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10(-/-) mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells.

RESULTS

Treatment of C3H.IL-10(-/-) mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-gamma and interleukin (IL)-17. The target for fenofibrate, PPARalpha, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10(-/-) mice, and fenofibrate repressed interferon-gamma and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, <em>CCL2</em>, and <em>CCL2</em>0, and repressed CXCL10 gene promoter activity in tumor necrosis factor-alpha-treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARdelta ligand GW0742 accelerated the onset of colitis in IL-10(-/-) mice.

CONCLUSIONS

The immunopathology observed in IL-10(-/-) mice resembles that seen in Crohn's disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn's disease.

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.

Pneumococcal disease continues to account for significant morbidity and mortality worldwide. For the development of novel prophylactic and therapeutic strategies against the disease spectrum, a complete understanding of pneumococcal behavior in vivo is necessary. We evaluated the expression patterns of the proven and putative virulence factor genes adcR, cbpA, cbpD, cbpG, cpsA, nanA, pcpA, piaA, ply, psaA, pspA, and spxB after intranasal infection of CD1 mice with serotype 2, 4, and 6A pneumococci by real-time reverse transcription-PCR. Simultaneous gene expression patterns of selected host immunomodulatory molecules, CCL2, CCL5, CD54, CXCL2, interleukin-6, and tomor necrosis factor alpha, were also investigated. We show that pneumococcal virulence genes are differentially expressed in vivo, with some genes demonstrating niche- and serotype-specific differential expression. The in vivo expression patterns could not be attributed to in vitro differences in expression of the genes in transparent and opaque variants of the three strains. The host molecules were significantly upregulated, especially in the lungs, blood, and brains of mice. The pneumococcal-gene expression patterns support their ascribed roles in pathogenesis, providing insight into which protein combinations might be more appropriate as vaccine antigens against invasive disease. This is the first simultaneous comparison of bacterial- and host gene expression in the same animal during pathogenesis. The strategy provides a platform for prospective evaluation of interaction kinetics between invading pneumococci and human patients in culture-positive cases and should be feasible in other infection models.

17β-estradiol (E) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. Our previous work demonstrates that both steroids compensate neuronal damage and activate distinct neuroprotective strategies such as improving local energy metabolism and abating pro-inflammatory responses. The current study explored steroid hormone-mediated protection from brain damage and restoration of behavioral function after 1h transient middle cerebral artery occlusion (tMCAO). Male and ovariectomized female rats were studied 24h after stroke. Both steroid hormones reduced the cortical infarct area in males and females to a similar extent. A maximum effect of ~60-70% reduction of the infarct size was evident after P and a combined treatment with both hormones. No infarct protection was seen in the basal ganglia. Testing of motor and sensory behavioral revealed an equal high degree of functional recovery in all three hormone groups. Gene expression studies in the delineated penumbra revealed that estrogen receptor (ER) alpha and beta are locally up-regulated. tMCAO-mediated induction of the pro-inflammatory chemokines CCL2, CCL5 and interleukin 6 was attenuated by E and P, whereas the expression of vascular endothelial growth factor (VEGF) was fortified. Local expression of microglia/macrophage/lymphocyte markers, i.e. Iba1, CD68 and CD3, were significantly reduced in the penumbra after hormone treatment suggesting attenuation of microglia and lymphocyte attraction. These results demonstrate the neuroprotective potency of a combined treatment with E and P under ischemic conditions in both sexes and point at the regulation of chemokine-microglia/lymphocyte interactions as a supposable mechanism implicated in cell protection.

Cytokines and chemokines are secreted, small cell-signaling protein molecules, whose receptors are expressed on immune cells. These factors play a critical role in immune cell differentiation, migration, and polarization into functional subtypes and in directing their biological functions. Much attention has been devoted to exploring the role of key inflammatory cytokines and promigratory chemokines in autoimmune, autoinflammatory, and allergic diseases, leading to development of therapeutic strategies that are based on their targeted neutralization. Recent studies, including those coming from our groups, show that several major proinflammatory cytokines and chemokines, including IFN-γ, IL-2, CCL2, and CXCL12, may also function as anti-inflammatory mediators and therefore, may have potential as anti-inflammatory drugs. Likewise, major anti-inflammatory mediators, such as TGF-β, may under certain conditions, in combination with other cytokines, exhibit proinflammatory function and direct the polarization of the highly inflammatory CD4(+) Th17 cells. We show here that the biological function of pro- and anti-inflammatory cytokines is dependent on three key parameters: the local concentration of a given cytokine, the stage of disease in which it is administered, and its combination with other cytokines. The therapeutic implications of these findings are discussed, including two very recent studies summarizing clinical trials, in which low-dose administration of IL-2 was used to successfully suppress HCV and GVHD.

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.

The aim of this study was to evaluate the long-term effects of lithium treatment on neonatal hypoxic-ischemic brain injury, inflammation, and neural stem/progenitor cell (NSPC) proliferation and survival. Nine-day-old male rats were subjected to unilateral hypoxia-ischemia (HI) and 2 mmol/kg lithium chloride was injected intraperitoneally immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-hour intervals for 7 days. Animals were killed 6, 24, 72 hours, or 7 weeks after HI. Lithium reduced total tissue loss by 69%, from 89.4±14.6 mm(3) in controls (n=15) to 27.6±6.2 mm(3) in lithium-treated animals (n=14) 7 weeks after HI (P<0.001). Microglia activation was inhibited by lithium treatment, as judged by Iba-1 and galectin-3 immunostaining, and reduced interleukin-1β and CCL2 levels. Lithium increased progenitor, rather than stem cell, proliferation in both nonischemic and ischemic brains, as judged by 5-bromo-2-deoxyuridine labeling 24 and 72 hours as well as by phospho-histone H3 and brain lipid-binding protein labeling 7 weeks after HI. Lithium treatment also promoted survival of newborn NSPCs, without altering the relative levels of neuronal and astroglial differentiation. In summary, lithium conferred impressive, morphological long-term protection against neonatal HI, at least partly by inhibiting inflammation and promoting NSPC proliferation and survival.

Several members of the chemokine family play an important role in reparative fibrosis and are involved in the pathogenesis of remodeling following myocardial infarction. Chemokines may regulate the fibrotic process through recruitment and activation of mononuclear cell subsets and fibroblast progenitors (fibrocytes), by exerting direct effects on resident fibroblasts, and by modulating angiogenesis. Monocyte Chemoattractant Protein (MCP)-1/CCL2 is the best studied chemokine in cardiac fibrosis. Disruption of the MCP-1 axis reduces fibrosis attenuating dilation of the infarcted ventricle. In addition, MCP-1 signaling is activated in response to insults that do not cause cardiomyocyte death, such as brief ischemia or pressure overload and regulates fibrous tissue deposition in experimental models of fibrotic non-infarctive cardiomyopathy. Understanding the role of chemokine-mediated interactions in the development of cardiac fibrosis may identify novel therapeutic targets for treatment of patients with heart failure.

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5' region close to the transcriptional start site and in the far 3' region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-kappaB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-kappaB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-kappaB and HDAC3 at the level of chromatin.

Cerebral inflammatory responses may initiate secondary cascades following traumatic brain injury (TBI). Changes in the expression of both cytokines and chemokines may activate, regulate, and recruit innate and adaptive immune cells associated with secondary degeneration, as well as alter a host of other cellular processes. In this study, we quantified the temporal expression of a large set of inflammatory mediators in rat cortical tissue after brain injury. Following a controlled cortical impact (CCI) on young adult male rats, cortical and hippocampal tissue of the injured hemisphere and matching contralateral material was harvested at early (4, 12, and 24 hours) and extended (3 and 7 days) time points post-procedure. Naïve rats that received only anesthesia were used as controls. Processed brain homogenates were assayed for chemokine and cytokine levels utilizing an electrochemiluminescence-based multiplex ELISA platform. The temporal profile of cortical tissue samples revealed a multi-phasic injury response following brain injury. CXCL1, IFN-γ, TNF-α levels significantly peaked at four hours post-injury compared to levels found in naïve or contralateral tissue. CXCL1, IFN-γ, and TNF-α levels were then observed to decrease at least 3-fold by 12 hours post-injury. IL-1β, IL-4, and IL-13 levels were also significantly elevated at four hours post-injury although their expression did not decrease more than 3-fold for up to 24 hours post-injury. Additionally, IL-1β and IL-4 levels displayed a biphasic temporal profile in response to injury, which may suggest their involvement in adaptive immune responses. Interestingly, peak levels of CCL2 and CCL2CCL2 levels in injured cortical tissue were significantly higher than peak levels of any other inflammatory mediator measured, thus suggesting a possible use as a biomarker. Fully elucidating chemokine and cytokine signaling properties after brain injury may provide increased insight into a number of secondary cascade events that are initiated or regulated by inflammatory responses.