Oxidatively modified low-density lipoprotein (oxLDL) plays a key role in the initiation of atherosclerosis by increasing monocyte adhesion. The mechanism that is responsible for the oxLDL-induced atherogenic monocyte recruitment in vivo, however, still remains unknown. Oxidation of LDL generates lysophosphatidylcholine, which is the main substrate for the lysophosphatidic acid (LPA) generating enzyme autotaxin. We show that oxLDL requires endothelial LPA receptors and autotaxin to elicit CXCL1-dependent arterial monocyte adhesion. Unsaturated LPA releases endothelial CXCL1, which is subsequently immobilized on the cell surface and mediates LPA-induced monocyte adhesion. Local and systemic application of LPA accelerates the progression of atherosclerosis in mice. Blocking the LPA receptors LPA(1) and LPA(3) reduced hyperlipidemia-induced arterial leukocyte arrest and atherosclerosis in the presence of functional CXCL1. Thus, atherogenic monocyte recruitment mediated by hyperlipidemia and modified LDL crucially depends on LPA, which triggers endothelial deposition of CXCL1, revealing LPA signaling as a target for cardiovascular disease treatments.

The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1beta, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1beta and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

OBJECTIVE

Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout.

METHODS

Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively.

RESULTS

Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1β. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome.

CONCLUSIONS

These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach.

Oligodendrocytes are cells that myelinate axons, providing saltatory conduction of action potentials and proper function of the central nervous system. Myelination begins prenatally in the human, and the sequence of oligodendrocyte development and the onset of myelination are not thoroughly investigated. This knowledge is important to better understand human diseases, such as periventricular leukomalacia, one of the leading causes of motor deficit in premature babies, and demyelinating disorders such as multiple sclerosis (MS). In this review we discuss the spatial and temporal progression of oligodendrocyte lineage characterized by the expression of specific markers and transcription factors in the human fetal brain from the early embryonic period (5 gestational weeks, gw) until midgestation (24 gw). Our in vitro evidence indicated that a subpopulation of human oligodendrocytes may have dorsal origin, from cortical radial glia cells, in addition to their ventral telencephalic origin. Furthermore, we demonstrated that the regulation of myelination in the human fetal brain includes positive and negative regulators. Chemokines, such as CXCL1, abundant in proliferative zones during brain development and in regions of remyelination in adult, are discussed in the view of their potential roles in stimulating oligodendrocyte development. Other signals are inhibitory and may include, but are not limited to, polysialic acid modification of the neural cell adhesion molecule on axons. Overall, important differences in temporal and spatial distribution and regulatory signals for oligodendrocyte differentiation exist between human and rodent brains. Those differences may underlie the unique susceptibility of humans to demyelinating diseases, such as MS.

OBJECTIVE

Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1(+) versus PD-L1(-) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1(+) melanoma microenvironment coordinately contributing to immunosuppression.

METHODS

Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1(+) versus PD-L1(-) melanomas.

RESULTS

Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1(+) melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1(+) melanomas, involved in CD8(+) T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells.

CONCLUSIONS

These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Islets from patients with type 2 diabetes exhibit β cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and <em>CXCL1</em> by islets and induced secretion of TNF-α, IL-1α, IL-1β, CCL2, CCL3, <em>CXCL1</em>, CXCL2, and <em>CXCL1</em>0 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to β cell dysfunction in type 2 diabetes and islet transplantation.

Exercise is a treatment paradigm that can ameliorate cognitive dysfunction in Alzheimer disease (AD) and AD mouse models. Since exercise is also known to alter the peripheral immune response, one potential mechanism for the cognitive improvement following exercise may be by modulating the inflammatory repertoire in the central nervous system. We investigated the effects of voluntary exercise in the Tg2576 mouse model of AD at a time-point at which pathology has already developed. Inflammatory mRNA markers are increased in sedentary Tg2576 mice versus non-transgenic controls. We demonstrate that short-term voluntary wheel running improved spatial learning in aged transgenic mice as compared to sedentary Tg2576 controls. Inflammatory profiles of the Tg2576 and non-transgenic mice were different following exercise with the non-transgenic mice showing a broader response as compared to the Tg2576. Notably, exercising Tg2576 exhibited increases in a few markers including <em>CXCL1</em> and <em>CXCL1</em>2, two chemokines that may affect cognition.

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

Accumulating evidence suggests that spinal cord astrocytes play an important role in neuropathic pain sensitization by releasing astrocytic mediators (e.g. cytokines, chemokines and growth factors). However, it remains unclear how astrocytes control the release of astrocytic mediators and sustain late-phase neuropathic pain. Astrocytic connexin-43 (now known as GJ1) has been implicated in gap junction and hemichannel communication of cytosolic contents through the glial syncytia and to the extracellular space, respectively. Connexin-43 also plays an essential role in facilitating the development of neuropathic pain, yet the mechanism for this contribution remains unknown. In this study, we investigated whether nerve injury could upregulate connexin-43 to sustain late-phase neuropathic pain by releasing chemokine from spinal astrocytes. Chronic constriction injury elicited a persistent upregulation of connexin-43 in spinal astrocytes for >3 weeks. Spinal (intrathecal) injection of carbenoxolone (a non-selective hemichannel blocker) and selective connexin-43 blockers (connexin-43 mimetic peptides (43)Gap26 and (37,43)Gap27), as well as astroglial toxin but not microglial inhibitors, given 3 weeks after nerve injury, effectively reduced mechanical allodynia, a cardinal feature of late-phase neuropathic pain. In cultured astrocytes, TNF-α elicited marked release of the chemokine CXCL1, and the release was blocked by carbenoxolone, Gap26/Gap27, and connexin-43 small interfering RNA. TNF-α also increased connexin-43 expression and hemichannel activity, but not gap junction communication in astrocyte cultures prepared from cortices and spinal cords. Spinal injection of TNF-α-activated astrocytes was sufficient to induce persistent mechanical allodynia, and this allodynia was suppressed by CXCL1 neutralization, CXCL1 receptor (CXCR2) antagonist, and pretreatment of astrocytes with connexin-43 small interfering RNA. Furthermore, nerve injury persistently increased excitatory synaptic transmission (spontaneous excitatory postsynaptic currents) in spinal lamina IIo nociceptive synapses in the late phase, and this increase was suppressed by carbenoxolone and Gap27, and recapitulated by CXCL1. Together, our findings demonstrate a novel mechanism of astrocytic connexin-43 to enhance spinal cord synaptic transmission and maintain neuropathic pain in the late-phase via releasing chemokines.

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1(-/-) mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of <em>CXCL1</em> and CXCL2 were increased in Cxcr2(-/-) mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3(-/-) mice revealed increased levels of circulating and brain <em>CXCL1</em>0 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1+ T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-gamma-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1+ cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.

The ability of an individual to sense pain is fundamental for its capacity to adapt to its environment and to avoid damage. The sensation of pain can be enhanced by acute or chronic inflammation. In the present study, we have investigated whether inflammatory pain, as measured by hypernociceptive responses, was modified in the absence of the microbiota. To this end, we evaluated mechanical nociceptive responses induced by a range of inflammatory stimuli in germ-free and conventional mice. Our experiments show that inflammatory hypernociception induced by carrageenan, lipopolysaccharide, TNF-alpha, IL-1beta, and the chemokine CXCL1 was reduced in germ-free mice. In contrast, hypernociception induced by prostaglandins and dopamine was similar in germ-free or conventional mice. Reduction of hypernociception induced by carrageenan was associated with reduced tissue inflammation and could be reversed by reposition of the microbiota or systemic administration of lipopolysaccharide. Significantly, decreased hypernociception in germ-free mice was accompanied by enhanced IL-10 expression upon stimulation and could be reversed by treatment with an anti-IL-10 antibody. Therefore, these results show that contact with commensal microbiota is necessary for mice to develop inflammatory hypernociception. These findings implicate an important role of the interaction between the commensal microbiota and the host in favoring adaptation to environmental stresses, including those that cause pain.

Epithelial cells provide the first line of defense against mucosal pathogens; however, their coordination with innate and adaptive immune cells is not well understood. Using mice with conditional gene deficiencies, we found that lymphotoxin (LT) from innate cells expressing transcription factor RORgammat, but not from adaptive T and B cells, was essential for the control of mucosal C. rodentium infection. We demonstrate that the LTbetaR signaling was required for the regulation of the early innate response against infection. Furthermore, we have revealed that LTbetaR signals in gut epithelial cells and hematopoietic-derived cells coordinate to protect the host from infection. We further determined that LTbetaR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1 and CXCL2 chemokines. These results support a model wherein LT from RORgammat(+) cells orchestrates the innate immune response against mucosal microbial infection.

Thymic stromal lymphopoietin (TSLP) is highly expressed by bronchial epithelial cells and skin keratinocytes in allergic diseases. TSLP acts as a master switch for allergic inflammation through the activation of dendritic cells and mast cells for initiating inflammatory type 2 T-helper lymphocyte responses. To elucidate the immunological cascades of epithelium/keratinocyte-eosinophil-mediated allergic inflammation, we examined the modulating effects of TSLP on human eosinophils. Expression of TSLP receptor complex was detected by RT-PCR, flow cytometry, and Western blot. Adhesion molecules, cytokine, and chemokines were quantitated by flow cytometry or ELISA. Intracellular signal transduction molecules were measured by Western blot and flow cytometry. We observed that human eosinophils constitutively expressed functional heterodimeric TSLP receptor complex comprising TSLP-binding chain TSLPR and IL-7Ralpha chain. TSLP could significantly delay eosinophil apoptosis, up-regulate cell surface expression of adhesion molecule CD18 and intercellular adhesion molecule-1, but down-regulate L-selectin, enhance eosinophil adhesion onto fibronectin, and induce the release of inflammatory cytokine IL-6 and chemokines CXCL8, CXCL1, and CCL2 (all P < 0.05). All these effects were concentration dependent and TSLP specific. TSLP regulated the above effects through the activation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase, and NF-kappaB signaling pathway, but not signal transducer and activator of transcription 5 and 3, which were usually activated in other effector cells upon TSLP stimulation. Collectively, the above findings elucidate the proallergic mechanisms of TSLP via the activation of distinct intracellular signaling pathways in eosinophils.

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset of vessels in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet alpha-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention.

Dendritic cells (DC) are believed to play an important role in the initiation of innate and adaptive immune responses to infection, including respiratory tract infections, where respiratory DC (RDC) perform this role. In this report, we examined the susceptibilities of isolated murine RDC to influenza virus infection in vitro and the effect of the multiplicity of infection (MOI) on costimulatory ligand upregulation and inflammatory cytokine/chemokine production after infection. We found that the efficiency of influenza virus infection of RDC increased with increasing MOIs. Furthermore, distinct subpopulations of RDC differed in their susceptibilities to influenza virus infection and in the magnitude/tempo of costimulatory ligand expression. Additional characterization of the CD11c-positive (CD11c(+)) RDC revealed that the identifiable subsets of RDC differed in susceptibility to infection, with CD11c(+) CD103(+) DC exhibiting the greatest susceptibility, CD11c(+) CD11b(hi) DC exhibiting intermediate susceptibility, and CD11c(+) B220(+) plasmacytoid DC (pDC) exhibiting the least susceptibility to infection. A companion analysis of the in vivo susceptibilities of these RDC subsets to influenza virus revealed a corresponding infection pattern. The three RDC subsets displayed different patterns of cytokine/chemokine production in response to influenza virus infection in vitro: pDC were the predominant producers of most cytokines examined, while CD103(+) DC and CD11b(hi) DC produced elevated levels of the murine chemokine CXCL1 (KC), interleukin 12p40, and RANTES in response to influenza virus infection. Our results indicate that RDC are targets of influenza virus infection and that distinct RDC subsets differ in their susceptibilities and responses to infection.

IL-17A and IL-17F are members of the IL-17 family that play crucial roles in allergic inflammation. Recent studies reported that IL-17A and IL-17F production from a distinct Th lymphocyte subset, Th17, was specifically induced by IL-23, which was produced by dendritic cells and macrophages in response to microbial stimuli. The IL-23-IL-17 axis might therefore provide a link between infections and allergic diseases. In the present study, we investigated the effects of IL-17A, IL-17F, and IL-23, alone or in combination, on cytokine and chemokine release from eosinophils and the underlying intracellular mechanisms. Human eosinophils were found to constitutively express receptors for IL-17A, IL-17F, and IL-23 at the protein level. IL-17A, IL-17F, and IL-23 could induce the release of chemokines GRO-alpha/CXCL1, IL-8/CXCL8, and MIP-1beta/CCL4 from eosinophils, while IL-17F and IL-23 could also increase the production of proinflammatory cytokines IL-1beta and IL-6. Synergistic effects were observed in the combined treatment of IL-17F and IL-23 on the release of proinflammatory cytokines, and the effects were dose-dependently enhanced by IL-23, but not IL-17F. Further investigations showed that IL-17A, IL-17F, and IL-23 differentially activated the ERK, p38 MAPK, and NF-kappaB pathways. Moreover, inhibition of these pathways using selective inhibitors could significantly abolish the chemokine release induced by IL-17A, IL-17F, and IL-23 and the synergistic increases on IL-1beta and IL-6 production mediated by combined treatment of IL-17F and IL-23. Taken together, our findings provide insight for the Th17 lymphocyte-mediated activation of eosinophils via differential intracellular signaling cascades in allergic inflammation.

IL-25 initiates, promotes, and augments Th2 immune responses. In this study, we report that Act1, a key component in IL-17-mediated signaling, is an essential signaling molecule for IL-25 signaling. Although Act1-deficient mice showed reduced expression of KC (CXCL1) and neutrophil recruitment to the airway compared with wild-type mice in response to IL-17 stimulation, Act1 deficiency abolished IL-25-induced expression of IL-4, IL-5, IL-13, eotaxin-1 (CCL11), and pulmonary eosinophilia. Using a mouse model of allergic pulmonary inflammation, we observed diminished Th2 responses and lung inflammation in Act1-deficient mice compared with wild-type mice. Importantly, Act1 deficiency in epithelial cells reduced the phenotype of allergic pulmonary inflammation due to loss of IL-17-induced neutrophilia and IL-25-induced eosinophilia, respectively. These results demonstrate the essential role of epithelial-derived Act1 in allergic pulmonary inflammation through the distinct impact of the IL-17R-Act1 and IL-25R-Act1 axes. Such findings are crucial for the understanding of pathobiology of atopic diseases, including allergic asthma, which identifies Act1 as a potential therapeutic target.

A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR(+) CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR(+) CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS.

Chemokines are important mediators in immune responses and inflammatory processes. Calcitonin gene-related peptide (CGRP) is produced in dorsal root ganglion (DRG) neurons. In this study, CGRP radioimmunoassay was used to investigate whether the chemokines CCL2 and CXCL1 could trigger CGRP release from cultured DRG neurons of neonatal rats and, if so, which cellular signaling pathway was involved. The results showed that CCL2 and CXCL1 ( approximately 5-100 ng/ml) evoked CGRP release and intracellular calcium elevation in a pertussis toxin (PTX)-sensitive manner. The CGRP release by CCL2 and CXCL1 was significantly inhibited by EGTA, omega-conotoxin GVIA (an N-type calcium channel blocker), thapsigargin, and ryanodine. Pretreatment of DRG neurons for 30 min with the inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not mitogen-activated protein kinases (MAPKs) significantly reduced CCL2- or CXCL1-induced CGRP release and intracellular calcium elevation. Intraplantar injection of CCL2 or CXCL1 produced hyperalgesia to thermal and mechanical stimulation in rats. These data suggest that CCL2 and CXCL1 can stimulate CGRP release and intracellular calcium elevation in DRG neurons. PLC-, PKC-, and calcium-induced calcium release from ryanodine-sensitive calcium stores signaling pathways are involved in CCL2- and CXCL1-induced CGRP release from primary nociceptive neurons, in which chemokines produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons.