BACKGROUND

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) share histopathological features but display different disease courses; we measured the concentration of 50 inflammatory mediators in the cerebrospinal fluid (CSF) of patients with either of these diseases.

METHODS

CSF samples were collected during a diagnostic lumbar puncture and stored at -30 degrees C. We analyzed the CSF of nine subjects with GBS; eight with CIDP; eight with diabetic polyneuropathy (DP) and seven with headache (controls). Fifty inflammatory mediators were simultaneously measured with a multiplex bead-based ELISA on a Suspension Array System. After Bonferroni's correction for repeated measures, non-parametric variance and post hoc test were calculated.

RESULTS

Thirty-two inflammatory mediators were expressed. The median concentration of IL-6, IL-9, IL-15, IL-18, CCL4, <em>CXCL1</em>, LIF, MIF, PDGFbb, IFN-gamma2, IL-2ra, IL-12(p40), IL-16, SCGF-b, TRAIL, FGF, G-CSF, GM-CSF, and M-CSF was not different among groups (variance: n.s.). The median concentration of CCL2, CCL7, CCL27, CXCL9, <em>CXCL1</em>0, <em>CXCL1</em>2, ICAM-1, VCAM1 and VEGF was higher in CIDP and GBS compared with controls (p<0.002). The median concentration of IL-8 and IL-1ra was higher in GBS than CIDP or DP or controls, whereas stem cell factor (SCF) and hepatocyte growth factor (HGF) were higher in CIDP than GBS or DP or controls (p<0.002).

CONCLUSIONS

Mediators of the recruitment and activation of lymphocytes and monocytes are expressed in the CSF of CIDP and GBS. IL-8 and IL-1ra are characteristic of GBS, whereas growth factors (SCF, HGF) of CIDP are possibly related to chronicity or to the survival/repair processes of neurons.

Non-CNS chemokine production may contribute to previously unrecognised components of Multiple Sclerosis (MS) pathology. Here we show that IL-8, a neutrophil chemoattractant, is significantly increased in serum from individuals with MS, and that the rodent homolog of IL-8 (CXCL1) is expressed in the liver in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. The hepatic expression of CXCL1 in EAE is accompanied by neutrophil recruitment to the liver, and we show that this recruitment is a feature of post mortem liver tissue from MS patients, which is a previously unrecognised phenomenon. We speculated that the presence of peripheral CXC-chemokine expression might contribute to the sickness behaviours associated with MS, which are a significant contributor to morbidity. Peripheral, but not central, administration of CXCL1 to Wistar rats inhibited spontaneous activity in the open field and burrowing behaviour in a dose-dependent manner (5-45 microg). The expression of CXCL1 by the liver and the recruitment of neutrophils can be modelled by the intracerebral injection of IL-1beta. Here, we found that interferon-beta (IFN-beta) pretreatment significantly inhibited hepatic CXCL1 production and neutrophil recruitment to the liver induced by the microinjection of IL-1beta into the brain. Thus while the mechanism by which IFN-beta therapy suppresses disease in MS remains unclear, the data presented here suggests that the inhibition of hepatic chemokine synthesis may be a contributing factor.

Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor, which exists both as membrane-anchored tumor necrosis factor and as cleaved soluble tumor necrosis factor forms. We previously demonstrated that tumor necrosis factor knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that tumor necrosis factor is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact membrane-anchored tumor necrosis factor, but no soluble tumor necrosis factor, display reduced infarct volumes at one and five days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of tumor necrosis factor and tumor necrosis factor-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1β, IL-6) or chemokines (CXCL1, CXCL1CXCL1 was reduced in membrane-anchored tumor necrosis factor(Δ/Δ) compared to membrane-anchored tumor necrosis factor(wt/wt) mice one day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of membrane-anchored tumor necrosis factor(Δ/Δ) mice. Collectively, these findings indicate that membrane-anchored tumor necrosis factor mediates the protective effects of tumor necrosis factor signaling in experimental stroke, and therapeutic strategies specifically targeting soluble tumor necrosis factor could be beneficial in clinical stroke therapy.

In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.

Endoplasmic reticulum disulfide oxidase ERO1-α plays a role in the formation of disulfide bonds in collaboration with protein disulfide isomerase. Disulfide bond formation is required for the proper conformation and function of secreted and cell surface proteins. We found that ERO1-α was overexpressed in a variety of tumor types; therefore, we examined its role in tumor growth. In BALB/c mice, knockdown of ERO1-α within 4T1 mouse mammary gland cancer (KD) cells caused retardation of in vivo tumor growth compared with tumor growth of scrambled control (SCR) cells. In contrast, when ERO1-α-overexpressed 4T1 (OE) cells were compared with mock control cells, OE cells showed augmented tumor growth. However, differences in tumor growth were not observed among four groups of nude mice, suggesting that expression of ERO1-α diminished antitumor immunity. We observed dense peritumoral granulocytic infiltrates in tumors of wild-type 4T1 and SCR cells but not KD cells, and these cells were identified as polymorphonuclear myeloid-derived suppressor cells (MDSCs). In addition, production of G-CSF and CXCL1/2, which have intramolecular disulfide bonds, from KD cells was significantly decreased compared with that from SCR cells. In contrast, OE cells produced a larger amount of these molecules than did mock cells. These changes were regulated at the posttranscriptional level. These results suggest that overexpression of ERO1-α in the tumor inhibits the T cell response by recruiting polymorphonuclear MDSCs via regulation of MDSC-prone cytokines and chemokines.

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-), NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1β, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis.

In pigs and other monogastric animal, the weaning phase is commonly accompanied by an increased susceptibility to gut disorders such as diarrhoea owing to the induction of an inflammatory process in the intestine during weaning. Given the unfavourable effects of intestinal inflammation on feed consumption, digestive capacity of the intestine and growth of animals, controlling intestinal inflammation is a reasonable approach for the maintenance of performance characteristics of livestock animals. Therefore, this study aimed to study the anti-inflammatory potential of a commercial polyphenol-rich grape seed (GS) and grape marc (GM) meal-based feed additive in a well-established in vitro intestinal epithelium model (polarized Caco-2 cells). The anti-inflammatory potential was evaluated by studying the effect of an ethanolic extract obtained from the GS and GM meal-based feed additive (GSGME) on the pro-inflammatory transcription factor NF-κB, which is considered to play a key role in the induction of weaning-associated intestinal inflammation. The highest non-cytotoxic concentrations of the ethanolic GSGME dose dependently reduced TNFα-induced NF-κB transactivation and decreased TNFα-induced mRNA levels of the NF-κB target genes IL-1β, IL-8, MCP-1 and CXCL1 in Caco-2 intestinal cells (p < 0.05). No effect of the ethanolic GSGME was observed on the cytoprotective Nrf2 pathway in Caco-2 cells as evidenced by an unaltered Nrf2 transactivation and unchanged mRNA levels of Nrf2 target genes, such as GPX-2, NQO1, CYP1A1 and UGT1A1. In conclusion, this study shows that an ethanolic GSGME exerts anti-inflammatory effects in intestinal cells under in vitro conditions. Thus, polyphenol-rich GSGM meal-based feed additives may be useful for the inhibition or prevention of inflammatory processes in the intestine of livestock animals, in particular during states with inappropriate NF-κB activation in the intestinal tissue, such as the weaning phase. Future studies are warranted to prove the in vivo anti-inflammatory potential of GSGM meal-based feed additives.

BACKGROUND

Inflammation is thought to play a role in ischemic acute kidney injury (AKI). We have demonstrated that macrophage and dendritic cell depletion, using liposome-encapsulated clodronate (LEC), is protective against ischemic AKI.

METHODS

To determine whether macrophages or dendritic cells or both play a role in ischemic AKI, we performed ischemic AKI in CD11b-DTR mice that have a diphtheria toxin (DT)-induced depletion of CD11b cells (macrophages) and CD11c-DTR mice that have a DT-induced depletion of CD11c cells (dendritic cells).

RESULTS

While LEC-treated animals had a significant functional protection from AKI, CD11b-DTR and CD11c-DTR mice were not protected against AKI despite a similar degree of renal macrophage and dendritic cell depletion. Proinflammatory cytokines are known to play a role in ischemic AKI. To determine the possible reasons for the lack of protection in CD11b-DTR and CD11c-DTR mice compared to LEC-treated mice, 32 cytokines/chemokines were measured in these mice. Of the cytokines/chemokines measured, IL-6, MCP-1, GMCSF, IL-1β and CXCL1 (also known as IL-8 in humans or KC in mice) showed significant differences in the LEC-treated, CD11b-DTR and CD11c-DTR mice. MCP-1 and CXCL1 (known mediators of AKI), and also GMCSF and IL-1β were increased in AKI and decreased in LEC-treated AKI but not AKI in CD11b-DTR or CD11c-DTR mice.

CONCLUSIONS

These findings suggest that LEC-mediated protection from AKI is not simply mediated by depletion of renal macrophage or dendritic cell subpopulations. Protection against AKI in LEC-treated compared to CD11b-DTR or CD11c-DTR mice may be partially explained by differences in proinflammatory cytokine profiles.

Autologous adipose tissue or adipose tissue with additive adipose-derived mesenchymal stem cells (ADSCs) is used in the breast reconstruction of breast cancer patients who undergo mastectomy. ADSCs play an important role in the angiogenesis and adipogenesis, which make it much better than other materials. However, ADSCs may promote residual tumor cells to proliferate or metastasize, and the mechanism is still not fully understood. In this study, we demonstrated that human ADSCs (hADSCs) could facilitate tumor cells growth after co-injection with MCF7 and ZR-75-30 breast cancer cells (BCCs) by promoting angiogenesis, but hADSCs showed limited effect on the growth of MDA-MB-231 BCCs. Intriguingly, compared with ZR-75-30 tumor cells, MCF7 tumor cells were more potentially promoted by hADSCs in the aspects of angiogenesis and proliferation. Consistent with this, cytokine and angiogenesis array analyses showed that after co-injection with hADSCs, the CXCL1 and CXCL8 concentration were significantly increased in MCF7 tumor, but only moderately increased in ZR-75-30 tumor and did not increase in MDA-MB-231 tumor. Furthermore, we found that CXCL1/8 were mainly derived from hADSCs and could increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs) by signaling via their receptors CXCR1 and CXCR2. A CXCR1/2-specific antagonist (SCH527123) attenuated the angiogenesis and tumor growth in vivo. Our findings suggest that CXCL1/8 secreted by hADSCs could promote breast cancer angiogenesis and therefore provide better understanding of safety concerns regarding the clinical application of hADSCs and suggestion in further novel therapeutic options. Stem Cells 2017;35:2060-2070.

Previous reports have shown that osteoblasts are mechano-sensitive. Low-intensity pulsed ultrasound (LIPUS) induces osteoblast differentiation and is an established therapy for bone fracture. Here we have examined how LIPUS affects inflammatory responses of osteoblasts to LPS. LPS rapidly induced mRNA expression of several chemokines including CCL2, <em>CXCL1</em>, and <em>CXCL1</em>0 in both mouse osteoblast cell line and calvaria-derived osteoblasts. Simultaneous treatment by LIPUS significantly inhibited mRNA induction of <em>CXCL1</em> and <em>CXCL1</em>0 by LPS. LPS-induced phosphorylation of ERKs, p38 kinases, MEK1/2, MKK3/6, IKKs, TBK1, and Akt was decreased in LIPUS-treated osteoblasts. Furthermore, LIPUS inhibited the transcriptional activation of NF-κB responsive element and Interferon-sensitive response element (ISRE) by LPS. In a transient transfection experiment, LIPUS significantly inhibited TLR4-MyD88 complex formation. Thus LIPUS exerts anti-inflammatory effects on LPS-stimulated osteoblasts by inhibiting TLR4 signal transduction.

BACKGROUND

Activation of the NLRP3 inflammasome in gout amplifies the inflammatory response and mediates further damage. In the current study, we assessed the therapeutic effect of OLT1177, an orally active NLRP3 inflammasome inhibitor that is safe in humans, in murine acute arthritis models.

METHODS

Zymosan or monosodium urate (MSU) crystals were injected intra-articularly (i.a.) into mouse knee joints to induce reactive or gouty arthritis. Joint swelling, articular cell infiltration, and synovial cytokines were evaluated 25 hours and 4 hours following zymosan or MSU challenge, respectively. OLT1177 was administrated intraperitoneally by oral gavage or in the food by an OLT1177-enriched diet.

RESULTS

OLT1177 reduced zymosan-induced joint swelling (p < 0.001), cell influx (p < 0.01), and synovial levels of interleukin (IL)-1β, IL-6, and chemokine (C-X-C motif) ligand 1 (CXCL1) (p < 0.05), respectively, when compared with vehicle-treated mice. Plasma OLT1177 levels correlated (p < 0.001) dose-dependently with reduction in joint inflammation. Treatment of mice with OLT1177 limited MSU crystal articular inflammation (p>> 0.0001), which was associated with decreased synovial IL-1β, IL-6, myeloperoxidase, and CXCL1 levels (p < 0.01) compared with vehicle-treated mice. When administrated orally 1 hour after MSU challenge, OLT1177 reduced joint inflammation, processing of IL-1β, and synovial phosphorylated c-Jun N-terminal kinase compared with the vehicle group. Mice were fed an OLT1177-enriched diet for 3 weeks and then challenged i.a. with MSU crystals. Joint swelling, synovial IL-1β, and expression of Nlrp3 and Il1b were significantly reduced in synovial tissues in mice fed an OLT1177-enriched diet when compared with the standard diet group.

CONCLUSIONS

Oral OLT1177 is highly effective in ameliorating reactive as well as gouty arthritis.

Cannabidiol (CBD) is a natural nonpsychotropic cannabinoid from marijuana (Cannabis sativa) with anti-epileptic and anti-inflammatory properties. Effect of CBD on naive immune system is not precisely understood. In this study, we observed that administering CBD into naive mice triggers robust induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the peritoneum, which expressed functional arginase 1, and potently suppressed T cell proliferation ex vivo. Furthermore, CBD-MDSC suppressed LPS-induced acute inflammatory response upon adoptive transfer in vivo. CBD-induced suppressor cells were comprised of CD11b(+)Ly6-G(+)Ly6-C(+) granulocytic and CD11b(+)Ly6-G(-)Ly6-C(+) monocytic subtypes, with monocytic MDSC exhibiting higher T cell-suppressive function. Induction of MDSC by CBD was markedly attenuated in Kit-mutant (Kit(W/W-v)) mast cell-deficient mice. MDSC response was reconstituted upon transfer of wild-type bone marrow-derived mast cells in Kit(W/W-v) mice, suggesting the key role of cKit (CD117) as well as mast cells. Moreover, mast cell activator compound 48/80 induced significant levels of MDSC in vivo. CBD administration in mice induced G-CSF, CXCL1, and M-CSF, but not GM-CSF. G-CSF was found to play a key role in MDSC mobilization inasmuch as neutralizing G-CSF caused a significant decrease in MDSC. Lastly, CBD enhanced the transcriptional activity of peroxisome proliferator-activated receptor γ in luciferase reporter assay, and PPAR-γ selective antagonist completely inhibited MDSC induction in vivo, suggesting its critical role. Together, the results suggest that CBD may induce activation of PPAR-γ in mast cells leading to secretion of G-CSF and consequent MDSC mobilization. CBD being a major component of Cannabis, our study indicates that marijuana may modulate or dysregulate the immune system by mobilizing MDSC.

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

Nod-like receptors are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two Nod-like receptor family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected intraperitoneally with thioglycollate, and then peritoneal neutrophils were isolated 4 hr after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonists, respectively. The level of cytokines [interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)] and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in wild-type (WT) neutrophils. Increased production of cytokines and chemokines with MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-κB and MAPK in WT neutrophils, but not in Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and Toll-like receptor (TLR) agonists (Pam3 CSK4 and lipopolysaccharide) exerted synergistic effects on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-α, CXCL1 and CCL2 in neutrophils in response to Gram-negative bacteria. Our findings suggest that the Nod2-Rip2 axis may contribute to the innate immune response of neutrophils against bacterial infection.

IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described.

In vivo MI was performed by coronary artery occlusion in the absence or presence of a neutralizing IL-17 antibody for blocking IL-17 actions in vivo. IL-17 signaling was also assessed in isolated primary cardiomyocytes by Western blot, mRNA expression and immunostaining.

Expression of IL-17A, IL-17F and the IL-17 receptor (IL-17RA) were all increased following MI. Expression of several IL-17 target genes, including Cxcl1, Cxcl2, IL-1β, iNOS and IL-6 was also upregulated following MI. In addition, IL-17A promoted the expression of Cxcl1 and IL-6 in isolated cardiomyocytes in a MAPK and PI(3)K-dependent manner. IL-17A and ischaemia/reperfusion (I/R) injury were found to have an additive effect on Cxcl1 expression, suggesting that IL-17 may enhance myocardial neutrophil recruitment during MI. Moreover, protein levels of both IL-17R and IL-17A were enhanced following in vivo MI. Finally, blocking IL-17 signaling in vivo reduced the levels of apoptotic cell death markers following in vivo MI.

These data imply that the expression of IL-17 cytokines and their receptor are elevated during myocardial I/R injury and may play a fundamental role in post infarct inflammatory and apoptotic responses.

We applied global gene expression arrays, quantitative real-time PCR, immunostaining, and functional assays to untangle the role of High Mobility Groups proteins (HMGs) in human osteoarthritis (OA)-affected cartilage. Bioinformatics analysis showed increased mRNA expression of Damage-Associated Molecular Patterns (DAMPs): HMGA, HMGB, HMGN, SRY, LEF1, HMGB1, MMPs, and HMG/RAGE-interacting molecules (spondins and S100A4, S100A10, and S100A11) in human OA-affected cartilage as compared with normal cartilage. HMGB2 was down-regulated in human OA-affected cartilage. Immunohistological staining identified HMGB1 in chondrocytes in the superficial cartilage. Cells of the deep cartilage and subchondral bone showed increased expression of HMGB1 in OA-affected cartilage. HMGB1 was expressed in the nucleus, cytosol, and extracellular milieu of chondrocytes in cartilage. Furthermore, HMGB1 was spontaneously released from human OA-affected cartilage in ex vivo conditions. The effects of recombinant HMGB1 was tested on human cartilage and chondrocytes in vitro. HMGB1 stimulated mRNA of 2 NFκB gene enhancers (NFκB1 and NFκB2), 16 CC and CXC chemokines (IL-8, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2, CCL5, CCL8, <em>CXCL1</em>, <em>CXCL1</em>0, CXCL2, CXCL3, and CXCL6) by ≥10-fold. Furthermore, HMGB1 and IL-1β and/or tumor necrosis factor α (but not HMGI/Y) also significantly induced inducible nitric oxide synthase, NO, and interleukin (IL)-8 production in human cartilage and chondrocytes. The recombinant HMGB1 utilized in this study shows properties that are similar to disulfide-HMGB1. The differential, stage and/or tissue-specific expression of HMGB1, HMGB2, and S100A in cartilage was associated with regions of pathology and/or cartilage homeostasis in human OA-affected cartilage. Noteworthy similarities in the expression of mouse and human HMGB1 and HMGB2 were conserved in normal and arthritis-affected cartilage. The multifunctional forms of HMGB1 and S100A could perpetuate damage-induced cartilage inflammation in late-stage OA-affected joints similar to sterile inflammation. The paracrine effects of HMGB1 can induce chemokines and NO that are perceived to change cartilage homeostasis in human OA-affected cartilage.

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.

The functions of gammadelta T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized alphabeta T cells. However, their conservation throughout evolution suggests that gammadelta T cells are biologically unique. In ruminants, gammadelta T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from alphabeta T cells. In fact, bovine WC1(+) gammadelta T cells can act as APC for alphabeta T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1(+) gammadelta and CD4(+) alphabeta T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific alphabeta and gammadelta T cell clones express many of the same genes, gammadelta T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1(+) gammadelta T cells when compared with CD4(+) alphabeta T cells selected from peripheral blood.

Fusobacterium nucleatum is implicated in accelerating colorectal cancer (CRC) and is found within metastatic CRC cells in patient biopsies. Here, we found that bacterial invasion of CRC cells and cocultured immune cells induced a differential cytokine secretion that may contribute to CRC metastasis. We used a modified galactose kinase markerless gene deletion approach and found that F. nucleatum invaded cultured HCT116 CRC cells through the bacterial surface adhesin Fap2. In turn, Fap2-dependent invasion induced the secretion of the proinflammatory cytokines IL-8 and CXCL1, which are associated with CRC progression and promoted HCT116 cell migration. Conditioned medium from F. nucleatum-infected HCT116 cells caused naïve cells to migrate, which was blocked by depleting CXCL1 and IL-8 from the conditioned medium. Cytokine secretion from HCT116 cells and cellular migration were attenuated by inhibiting F. nucleatum host-cell binding and entry using galactose sugars, l-arginine, neutralizing membrane protein antibodies, or fap2 deletion. F. nucleatum also induces the mobilization of immune cells in the tumor microenvironment. However, in neutrophils and macrophages, the bacterial-induced secretion of cytokines was Fap2 independent. Thus, our findings show that F. nucleatum both directly and indirectly modulates immune and cancer cell signaling and migration. Because increased IL-8 and CXCL1 production in tumors is associated with increased metastatic potential and cell seeding, poor prognosis, and enhanced recruitment of tumor-associated macrophages and fibroblasts, we propose that inhibition of host-cell binding and invasion, potentially through vaccination or novel galactoside compounds, could be an effective strategy for reducing F. nucleatum-associated CRC metastasis.

It is clear that CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells inhibit chronic inflammatory responses as well as adaptive immune responses. Among the CD4(+) T-cell population in the skin, at least one-fifth express Foxp3. As the skin is constantly exposed to antigenic challenge and is a common site of vaccination, understanding the role of these skin-resident Treg cells is important. Although the suppressive effect of Treg cells on T cells is well documented, less is known about the types of innate immune cells influenced by Treg cells and whether the Treg cells suppress acute innate immune responses in vivo. To address this we used a mouse melanoma cell line expressing Fas ligand (B16FasL), which induces an inflammatory response following subcutaneous injection of mice. We demonstrate that Treg cells limit this response by inhibiting neutrophil accumulation and survival within hours of tumour cell inoculation. This effect, which was associated with decreased expression of the neutrophil chemoattractants CXCL1 and CXCL2, promoted survival of the inoculated tumour cells. Overall, these data imply that Treg cells in the skin are rapidly mobilized and that this activity serves to limit the amplification of inflammatory responses at this site.

Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis-induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β.

We have previously demonstrated that the thiazolidinedione ciglitazone inhibited, independently of PPARγ activation, melanoma cell growth. Further investigations now show that ciglitazone effects are mediated through the regulation of secreted factors. Q-PCR screening of several genes involved in melanoma biology reveals that ciglitazone inhibits expression of the CXCL1 chemokine gene. CXCL1 is overexpressed in melanoma and contributes to tumorigenicity. We show that ciglitazone induces a diminution of CXCL1 level in different human melanoma cell lines. This effect is mediated by the downregulation of microphthalmia-associated transcription factor, MITF, the master gene in melanocyte differentiation and involved in melanoma development. Further, recombinant CXCL1 protein is sufficient to abrogate thiazolidinedione effects such as apoptosis induction, whereas extinction of the CXCL1 pathway mimics phenotypic changes observed in response to ciglitazone. Finally, inhibition of human melanoma tumor development in nude mice treated with ciglitazone is associated with a strong decrease in MITF and CXCL1 levels. Our results show that anti-melanoma effects of thiazolidinediones involve an inhibition of the MITF/CXCL1 axis and highlight the key role of this specific pathway in melanoma malignancy.