PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

CXCL1 inhibits airway smooth muscle cell migration through the decoy receptor Duffy antigen receptor for chemokines.

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway.

ERK2-dependent activation of c-Jun is required for nontypeable Haemophilus influenzae-induced CXCL2 upregulation in inner ear fibrocytes.

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.

ICAM-1-mediated leukocyte adhesion is critical for the activation of endothelial LSP1.

Leukocyte-endothelial interaction triggers signaling events in endothelial cells prior to transendothelial migration of leukocytes. Leukocyte-specific protein 1 (LSP1), expressed in endothelial cells, plays a pivotal role in regulating subsequent recruitment steps following leukocyte adhesion. In neutrophils, LSP1 is activated by phosphorylation of its serine residues by molecules downstream of p38 MAPK and PKC. Whether leukocyte adhesion to endothelial cells is required for endothelial LSP1 activation remains elusive. In addition, discrepancies in the functions of endothelial and leukocyte LSP1 in leukocyte adhesion prevail. We demonstrate that adhesion of wild-type (Lsp1(+/+)) neutrophils to LSP1-deficient (Lsp1(-/-)) endothelial cells was significantly reduced compared with adhesion to Lsp1(+/+) endothelial cells. Immunoblotting revealed increased phosphorylated endothelial LSP1 in the presence of adherent Lsp1(-/-) neutrophils [stimulated by macrophage inflammatory protein-2 (CXCL2), TNF-α, or thapsigargin], but not cytokine or chemokine alone. Pharmacological inhibition of p38 MAPK by SB-203580 (10 µM) significantly blunted the phosphorylation of endothelial LSP1. Functionally blocking endothelial ICAM-1 or neutrophil ß2-integrins diminished neutrophil adhesion and phosphorylation of endothelial LSP1. The engagement of endothelial ICAM-1 cross-linking, which mimics leukocyte adhesion, resulted in phosphorylation of endothelial LSP1. In neutrophil-depleted Lsp1(+/+) mice, administration of ICAM-1 cross-linking antibody resulted in increased phosphorylation of LSP1 and p38 MAPK in TNF-α-stimulated cremaster muscle. In conclusion, endothelial LSP1 participates in leukocyte adhesion in vitro, and leukocyte adhesion through ICAM-1 fosters the activation of endothelial LSP1, an effect at least partially mediated by the activation of p38 MAPK. Endothelial LSP1, in contrast to neutrophil LSP1, is not phosphorylated by cytokine or chemokine stimulation alone.

Regulation of the estrous cycle by neutrophils via opioid peptides.

We found previously that neutrophil-depleted mice exhibited significant blockading of both the regular estrous cycle and cyclic changes of steroid hormone levels. In this study, we aimed at elucidation of the underlying mechanism. To examine the possibility that an increase in bacteria in the vaginal vault of neutrophil-depleted mice causes blockading of the estrous cycle, we treated neutrophil-depleted mice with antibiotics but failed to restore the estrous cycle. We then examined another possibility that neutrophils regulate the estrous cycle via opioid peptides, because opioid peptides regulate steroidogenesis in theca and granulosa cells in the ovaries, and because neutrophils contain opioid peptides. In support of this possibility, naloxone, an opioid antagonist, blocked the estrous cycle and a µ opioid receptor agonist restored the estrous cycle in neutrophil-depleted mice. Pro-opiomelanocortin was immunohistochemically detected in peripheral blood neutrophils but not in ones that had infiltrated into the ovaries. i.v. injection of anti-MIP-2 polyclonal Ab caused blockading of the estrous cycle, whereas MIP-2 was detected in the ovaries, suggesting a role of MIP-2 in the regulation of the estrous cycle. Moreover, i.v. injection of MIP-2 decreased the pro-opiomelanocortin signal in peripheral blood neutrophils and caused blockading of the estrous cycle. Together, these results suggest that neutrophils maintain the estrous cycle via opioid peptides.

Monocyte chemotactic protein-1 promotes inflammatory vascular repair of murine carotid aneurysms via a macrophage inflammatory protein-1α and macrophage inflammatory protein-2-dependent pathway.

BACKGROUND: Up to 5% of the population may have a brain aneurysm. If the brain aneurysm ruptures, there is >50% mortality, and more than one third of survivors are dependent. Brain aneurysms detected before rupture can be treated to prevent rupture, or ruptured aneurysms can be treated to prevent rerupture. Endovascular coiling of brain aneurysms is the treatment of choice for some aneurysms; however, up to one quarter of aneurysms may recur. The coiled aneurysms that do not recur are characterized by inflammatory intra-aneurysmal tissue healing; therefore, we studied the biology of this process, specifically the role of monocyte chemotactic protein-1 (MCP-1), a cytokine known for tissue healing. METHODS AND RESULTS: We created coils with a 50:50 poly-dl-lactic glycolic acid (PLGA) coating that released MCP-1 at 3 different doses (100 µg/mL, 1 mg/mL, and 10 mg/mL) and performed a dose-response study for effect on intra-aneurysmal tissue healing in a murine carotid aneurysm model. We then demonstrated that MCP-1 (100 µg/mL)-releasing coils promote significantly greater aneurysm tissue in-growth than bare platinum or PLGA-only coils. We show that MCP-1 recruits the migration of fibroblasts, macrophages, smooth muscle cells, and endothelial cells in vitro in cell migration assays and in vivo in murine carotid aneurysms. Using gfp(+) bone marrow-transplant chimeric mice, we demonstrate that the MCP-1-recruited fibroblasts and macrophages are derived from the bone marrow. We demonstrate that this MCP-1-mediated vascular inflammatory repair occurs via a macrophage inflammatory protein (MIP)-1α- and MIP-2-dependent pathway. MCP-1 released from coiled murine aneurysms causes significant upregulation of MIP-1α and MIP-2 expression by cytokine array assay. Blocking MIP-1α and MIP-2 with antagonist antibody causes a significant decrease in MCP-1-mediated intra-aneurysmal tissue healing. CONCLUSION: Our findings suggest that MCP-1 has a critical role in promoting inflammatory intra-aneurysmal tissue healing in an MIP-1α- and MIP-2-dependent pathway.

Different microvascular permeability responses elicited by the CXC chemokines MIP-2 and KC during leukocyte recruitment: role of LSP1.

The CXC chemokines keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein-2 (MIP-2, CXCL2) activate G-protein coupled receptor CXCR2 and are believed to have similar inflammatory effects in mice. Their specific signaling mechanisms remain elusive. A wide variety of cellular events, mediators and signaling pathways are known to regulate microvascular permeability. Leukocyte-specific protein 1 (LSP1), a Ca2+- and F-actin binding protein, is one of the major downstream substrates of p38 MAPK. LSP1 was previously shown to play a pivotal role in leukocyte transmigration and microvascular permeability. Using intravital microscopy visualizing microvasculature of murine cremaster muscle, we demonstrate that KC and MIP-2 triggered increased leukocyte recruitment which was significantly reduced in LSP1-deficient mice compared to the wild-type control mice. Fluorescence imaging revealed that KC induced more substantial increases of microvascular permeability to FITC-labeled albumin than MIP-2. We found that LSP1 had a more prominent role in microvascular hyperpermeability induced by KC than that triggered by MIP-2. Moreover, Western blotting showed enhanced phosphorylation of p38 MAPK in the cremasteric tissue after stimulation with KC but not with MIP-2 and KC-induced but not MIP-2-induced hyperpermeability was blunted by pharmacological inhibition of p38 MAPK. In conclusion, LSP1 plays an important role in leukocyte recruitment induced by both KC and MIP-2. KC elicits more profoundly increased microvascular permeability than MIP-2. KC is at least partially effective through LSP1 and the phosphorylation of p38 MAPK.

Epigenetic augmentation of the macrophage inflammatory protein 2/C-X-C chemokine receptor type 2 axis through histone H3 acetylation in injured peripheral nerves elicits neuropathic pain.

Although there is growing evidence showing that the involvement of chemokines in the pathogenesis of neuropathic pain is associated with neuroinflammation, the details are unclear. We investigated the C-X-C chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)]/C-X-C chemokine receptor type 2 (CXCR2) axis and epigenetic regulation of these molecules in neuropathic pain after peripheral nerve injury. Expression of MIP-2 and CXCR2 were up-regulated and localized on accumulated neutrophils and macrophages in the injured sciatic nerve (SCN) after partial sciatic nerve ligation (PSL). Perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea (SB225002) prevented PSL-induced tactile allodynia and thermal hyperalgesia. Perineural injection of recombinant MIP-2 elicited neuropathic pain-like behaviors. Anti-MIP-2 suppressed neutrophil accumulation in the SCN after PSL. Neutrophil depletion by intraperitoneal injection of Ly6G antibody attenuated PSL-induced neuropathic pain. Both anti-MIP-2 and SB225002 suppressed up-regulation of inflammatory cytokines and chemokines in the injured SCN. In addition, acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the promoter region of MIP-2 and CXCR2 was increased in the injured SCN after PSL. Expression of AcK9-H3 was observed in the nuclei of neutrophils and macrophages surrounding the epineurium. Administration of the histone acetyltransferase inhibitor anacardic acid suppressed the up-regulation of MIP-2 and CXCR2 in the SCN after PSL and resulted in the prevention of PSL-induced neuropathic pain. Taken together, these results show that augmentation of the MIP-2/CXCR2 axis by hyperacetylation of histone H3 on the promoter region of MIP-2 and CXCR2 located in the injured peripheral nerve elicits chronic neuroinflammation through neutrophil accumulation, leading to neuropathic pain.

CXC chemokine signaling in the liver: impact on repair and regeneration.

The process of liver repair and regeneration following hepatic injury is complex and relies on a temporally coordinated integration of several key signaling pathways. Pathways activated by members of the CXC family of chemokines play important roles in the mechanisms of liver repair and regeneration through their effects on hepatocytes. However, little is known about the signaling pathways used by CXC chemokine receptors in hepatocytes. Here we review our current understanding of the pathways involved in both CXC chemokine receptor signaling in other cell types, most notably neutrophils, and similar pathways operant during hepatocyte proliferation/liver regeneration to formulate a basis for the function of CXC chemokine receptor signaling in hepatocytes.

CXC chemokine ligand 2 induced by receptor activator of NF-kappa B ligand enhances osteoclastogenesis.

CXCL2 has been known to regulate immune functions mainly by chemo-attracting neutrophils. In this study, we show that CXCL2 can be induced by receptor activator of NF-kappaB ligand, the osteoclast (OC) differentiation factor, through JNK and NF-kappaB signaling pathways in OC precursor cells. CXCL2 in turn enhanced the proliferation of OC precursor cells of bone marrow-derived macrophages (BMMs) through the activation of ERK. Knockdown of CXCL2 inhibited both the proliferation of and the ERK activation in BMMs. During osteoclastogenesis CXCL2 stimulated the adhesion and the migration of BMMs. Moreover, the formation of OCs from BMMs was significantly increased on treatment with CXCL2. Conversely, the CXCL2 antagonist repertaxin and a CXCL2 neutralizing Ab potently reduced receptor activator of NF-kappaB ligand-induced osteoclastogenesis. Furthermore, CXCL2 evoked fulminant bone erosion in the in vivo mouse experiments. Finally, prominent upregulation of CXCL2 was detected in synovial fluids and sera from rheumatoid arthritis patients, suggesting a potential involvement of CXCL2-mediated osteoclastogenesis in rheumatoid arthritis-associated bone destruction. Thus, CXCL2 is a novel therapeutic target for inflammatory bone destructive diseases.

Dysregulated macrophage-inflammatory protein-2 expression drives illness in bacterial superinfection of influenza.

Influenza virus infection is a leading cause of death and disability throughout the world. Influenza-infected hosts are vulnerable to secondary bacterial infection, however, and an ensuing bacterial pneumonia is actually the predominant cause of influenza-attributed deaths during pandemics. A number of mechanisms have been proposed by which influenza may predispose to superinfection with an unrelated or heterologous pathogen, but the subsequent interaction between the host, virus, and bacteria remains an understudied area. In this study, we develop and examine a novel model of heterologous pulmonary infection in which an otherwise subclinical Bordetella parapertussis infection synergizes with an influenza virus infection to yield a life-threatening secondary pneumonia. Despite a profound pulmonary inflammatory response and unaltered viral clearance, bacterial clearance was significantly impaired in heterologously infected mice. No deficits were observed in pulmonary or systemic adaptive immune responses or the viability or function of infiltrating inflammatory cells to explain this phenomenon, and we provide evidence that the onset of severe pulmonary inflammation actually precedes the increased bacterial burden, suggesting that exacerbated inflammation is independent of bacterial burden. To that end, neutralization of the ELR(+) inflammatory chemokine MIP-2 (CXCL2/GRO-beta) attenuated the inflammation, weight loss, and clinical presentation of heterologously infected mice without impacting bacterial burden. These data suggest that pulmonary inflammation, rather than pathogen burden, is the key threat during bacterial superinfection of influenza and that selective chemokine antagonists may be a novel therapeutic intervention in cases of bacterial superinfection of influenza.

C-X-C Chemokines Influence Intraocular Inflammation During Bacillus Endophthalmitis.

Purpose: The purpose of this study was to explore the C-X-C chemokines CXCL2 and CXCL10 as potential anti-inflammatory targets for Bacillus endophthalmitis. Methods: Bacillus endophthalmitis was induced in C57BL/6J, CXCL2-/-, and CXCL10-/- mice. At specific times postinfection, eyes were analyzed for Bacillus, retinal function, and inflammation. The efficacies of intravitreal anti-CXCL2 and anti-CXCL10 with or without gatifloxacin in B. cereus endophthalmitis were also assessed using the same techniques. Results: Despite similar Bacillus growth in eyes of C57BL/6J, CXCL2-/-, and CXCL10-/- mice, retinal function retention was greater in eyes of CXCL2-/- and CXCL10-/- mice compared to that of C57BL/6J mice. Neutrophil migration into eyes of CXCL2-/- and CXCL10-/- mice was reduced to a greater degree compared to that of eyes of C57BL/6J mice. Infected CXCL2-/- and CXCL10-/- mouse eyes had significantly less inflammation compared to that of C57BL/6J eyes. Retinal structures in infected eyes of CXCL2-/- mice were preserved for a longer time than in CXCL10-/- eyes. Compared to untreated eyes, there was less inflammation and significant retention of retinal function in eyes treated with anti-CXCL2 and anti-CXCL10 with or without gatifloxacin. Conclusions: For Bacillus endophthalmitis, the absence of CXCL2 or CXCL10 in mice resulted in retained retinal function and less inflammation. The absence of CXCL2 led to a better clinical outcome than the absence of CXCL10. The use of anti-CXCL2 and anti-CXCL10 limited inflammation during B. cereus endophthalmitis. These results highlight the utility of CXCL2 and CXCL10 as potential targets for anti-inflammatory therapy that can be tested in conjunction with antibiotics for improving treating Bacillus endophthalmitis.

Lipopolysaccharide promotes early endometrial-peritoneal interactions in a mouse model of endometriosis.

PURPOSE: The aims of this study were to clarify the effects of lipopolysaccharide (LPS) on the early development of endometriosis and on the production of cytokines and chemokines in the murine peritoneal cavity. METHODS: Endometriotic lesions were induced in C57BL/6J adult female mice by intraperitoneal injection of endometrial fragments plus blood or endometrial fragments plus blood with LPS. On day 7, endometriotic lesions were assessed by gross and microscopic evaluations. Time-dependent changes in the secretion of TNF-α,IL-6,and CXCL2/MIP-2 in peritoneal lavage fluid after the intraperitoneal injection of LPS (50 µg/body) were measured by their respective enzyme-linked immunosorbent assays. RESULTS: The areas of endometriotic lesions in the LPS group (10.8 8.6 mm2) were significantly larger than those in the control group (3.1 3.7 mm2).The levels of TNF-α and IL-6 peaked within 2 hours and the level of MIP-2 reached a maximum on day 1 after the injection of LPS. CONCLUSIONS: LPS promotes development of the early stages of murine endometriotic lesions. J. Med. Invest. 66 : 70-74, February, 2019.

Roles of chemokines in renal ischemia/reperfusion injury.

Ischemia-reperfusion injury (IR) is a common and an important clinical cause of renal disease, such as renal transplantation, renal artery stenosis and following shock from any cause. Inflammatory reaction after IR is regulated by various kinds of mediators. Chemokines are major mediators of the inflammation, and regulate pro-inflammatory cytokine and adhesion molecule expression, and leukocyte infiltration and activation. Chemokines are the key players of inflammation, angiogenesis and fibrosis. These inflammatory processes mediated by chemokines were observed in not only experimental animal models, but also in human renal diseases with ischemic injury. A number of challenges of chemokine targeted therapy is trying to prevent the ischemic injury, and will give some beneficial effect on the injury.

Chemokines and chemokine receptors in stem cell circulation.

Stem cells are rare, pluripotent, self-renewing cells that give rise to all mature cells during development and adult life. Due to their proliferative capabilities and their ability to home and contribute to the regeneration of damage tissue, stem cells can be transformed into established tumors. Stem cells can function as a double-edged sword--they have the ability to circulate and migrate throughout the developing and mature adult organism, which is essential for their normal function; however, transformed stem cells are also endowed with the machinery to metastasize into various organs. Chemokine and chemokine receptors play a critical role in directing the trafficking of these cells. It is therefore evident that understanding the role of chemokines and their receptors in stem cell circulation is critical for the successful use of these cells in therapy for a wide variety of pathological conditions.

Chemokines and neuromodulation.

Chemokines are not only mediators of the immune system and expressed in inflammatory situations. They are also constitutively expressed in the brain in both glial cells and neurons. Several recent evidence suggest that they can have a neurotransmitter/neuromodulatory role on brain functions similar to several neuropeptides reported so far. The aim of this short review is to illustrate that point using two chemokine systems, SDF-1/CXCL12 and its receptor CXCR4 and MCP-1/CCL2 and its receptor CCR2.

IL-21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration.

BACKGROUND AND PURPOSE: The immune system plays an important role in driving the acute inflammatory response following myocardial ischaemia/reperfusion injury (MIRI). IL-21 is a pleiotropic cytokine with multiple immunomodulatory effects, but its role in MIRI is not known. EXPERIMENTAL APPROACH: Myocardial injury, neutrophil infiltration and the expression of neutrophil chemokines KC (CXCL1) and MIP-2 (CXCL2) were studied in a mouse model of MIRI. Effects of IL-21 on the expression of KC and MIP-2 in neonatal mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were determined by real-time PCR and ELISA. The signalling mechanisms underlying these effects were explored by western blot analysis. KEY RESULTS: IL-21 was elevated within the acute phase of murine MIRI. Neutralization of IL-21 attenuated myocardial injury, as illustrated by reduced infarct size, decreased cardiac troponin T levels and improved cardiac function, whereas exogenous IL-21 administration exerted opposite effects. IL-21 increased the infiltration of neutrophils and increased the expression of KC and MIP-2 in myocardial tissue following MIRI. Moreover, neutrophil depletion attenuated the IL-21-induced myocardial injury. Mechanistically, IL-21 increased the production of KC and MIP-2 in neonatal CMs and CFs, and enhanced neutrophil migration, as revealed by the migration assay. Furthermore, we demonstrated that this IL-21-mediated increase in chemokine expression involved the activation of Akt/NF-κB signalling in CMs and p38 MAPK/NF-κB signalling in CFs. CONCLUSIONS AND IMPLICATIONS: Our data provide novel evidence that IL-21 plays a pathogenic role in MIRI, most likely by promoting cardiac neutrophil infiltration. Therefore, targeting IL-21 may have therapeutic potential as a treatment for MIRI. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-X-C motif) ligand 2 (CXCL2) modulate the activity of TRPV1+/IB4+ cultured rat dorsal root ganglia neurons upon short-term and acute application.

CXCL1 and CXCL2 are two chemokines with 78% homology of their sequence. CXCL1 was associated with atopic dermatitis, a highly pruritic skin disease, but it is not clear what is its mechanism of action, while for CXCL2 there are no data about an association with itch sensitivity. CXCL1 and CXCL2 can modulate TRPV1 receptors, which are one of the most important downstream effectors for itch sensitivity, upon short-term (4 h) or long-term (24 h) incubation, but the data are incomplete. Therefore, the aims of this study were to better characterize the short-term effects of CXCL1 and CXCL2 on TRPV1+/IB4+ dorsal root ganglia neurons known to include nociceptor and itch-sensitive neurons, and to obtain new data about the acute application (12 min) of the two chemokines on the same population of neurons. The results showed that 4 nM CXCL1 and 3.6 nM CXCL2 significantly reduce TRPV1 desensitization in TRPV1+/IB4+ DRG +neurons after short-term incubation, but when acutely applied CXCL1 activated a sub-population of itch-sensitive TRPV1+/IB4+ cells in a slow, low amplitude manner, while CXCL2 had a similar effect but on non-itch TRPV1+/IB4+ DRG neurons. These data contribute to a better understanding of CXCL1 and CXCL2 mechanism of action for both pain and itch inducing effects.

Site-Specific Neutrophil Migration and CXCL2 Expression in Periodontal Tissue.

The oral microbial community is the best-characterized bacterial ecosystem in the human host. It has been shown in the mouse that oral commensal bacteria significantly contribute to clinically healthy periodontal homeostasis by influencing the number of neutrophils that migrate from the vasculature to the junctional epithelium. Furthermore, in clinically healthy tissue, the neutrophil response to oral commensal bacteria is associated with the select expression of the neutrophil chemokine CXCL2 but not CXCL1. This preliminary study examined the contribution of commensal bacteria on neutrophil location across the tooth/gingival interface. Tissue sections from the root associated mesial (anterior) of the second molar to the root associated distal (posterior) of the second molar were examined for neutrophils and the expression of the neutrophil chemokine ligands CXCL1 and CXCL2. It was found that both the number of neutrophils as well as the expression of CXCL2 but not CXCL1 was significantly increased in tissue sections close to the interdental region, consistent with the notion of select tissue expression patterns for neutrophil chemokine expression and subsequent neutrophil location. Furthermore, mice gavaged with either oral Streptococcus or Lactobacillus sp. bacteria induced a location pattern of neutrophils and CXCL2 expression similar to the normal oral flora. These data indicate for the first time select neutrophil location and chemokine expression patterns associated with clinically healthy tissue. The results reveal an increased inflammatory load upon approaching the interproximal region, which is consistent with the observation that the interproximal region often reveals early clinical signs of periodontal disease.

The role of MyD88- and TRIF-dependent signaling in monophosphoryl lipid A-induced expansion and recruitment of innate immunocytes.

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.