MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

Monocyte chemotactic protein (MCP)-1 (CCL2) and its receptor (CCR2) are elevated in chronic heart failure facilitating lung monocyte infiltration and differentiation which may contribute to lung fibrosis.

BACKGROUND: Dyspnea, the cardinal manifestation of chronic heart failure (CHF), may reflect both pulmonary oedema and pulmonary remodeling resulting in tissue stiffening. Emerging evidence suggests that predominance of distinct phenotypes of alveolar and recruited macrophages, designated M1 and M2, may regulate the course of inflammatory tissue repair and remodeling in the lung. METHODS: In a CHF rat model, we found fibrotic reinforcement of the extracellular matrix with an increase in monocyte chemotactic protein (MCP)-1/CCL2 in bronchoalveolar lavage (BAL), corresponding to a 3-fold increase in recruited macrophages. In this clinical cross sectional study, we aimed to examine potential mediators of leukocyte activation and lung infiltration in parallel BAL and blood from CHF patients compared to non-CHF controls. RESULTS: Mini-BAL and peripheral blood samples were obtained from hospitalized CHF, acute decompensated CHF and non-CHF patients. CHF patients and decompensated CHF patients demonstrated increases from non-CHF patients in BAL MCP-1, as well as the M2 macrophage cytokines interleukin-10 and transforming growth factor-ß. BAL and plasma MCP-1 were significantly correlated; however, MCP-1 was 20-fold higher in epithelial lining fluid in BAL, indicative of an alveolar chemotactic gradient. An increase in transglutaminase 2 positive M2 macrophages in parallel with a decrease in the MCP-1 receptor, CC chemokine receptor 2 (CCR2), was apparent in BAL cells of CHF patients compared to non-CHF. CONCLUSION: These data suggest a pathway of MCP-1 mediated M2 macrophage prevalence in the lungs of CHF patients which may contribute to pulmonary fibrotic remodeling and consequent increased severity of dyspnea.

LncRNA MRF drives the regulatory function on monocyte recruitment and polarization through HNRNPD-MCP1 axis in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

Macrophage-Specific MCPIP1/Regnase-1 Attenuates Kidney Ischemia-Reperfusion Injury by Shaping the Local Inflammatory Response and Tissue Regeneration.

Sterile inflammation either resolves the initial insult or leads to tissue damage. Kidney ischemia/reperfusion injury (IRI) is associated with neutrophilic infiltration, enhanced production of inflammatory mediators, accumulation of necrotic cells and tissue remodeling. Macrophage-dependent microenvironmental changes orchestrate many features of the immune response and tissue regeneration. The activation status of macrophages is influenced by extracellular signals, the duration and intensity of the stimulation, as well as various regulatory molecules. The role of macrophage-derived monocyte chemoattractant protein-induced protein 1 (MCPIP1), also known as Regnase-1, in kidney ischemia-reperfusion injury (IRI) and recovery from sterile inflammation remains unresolved. In this study, we showed that macrophage-specific Mcpip1 deletion significantly affects the kidney phenotype. Macrophage-specific Mcpip1 transgenic mice displayed enhanced inflammation and loss of the tubular compartment upon IRI. We showed that MCPIP1 modulates sterile inflammation by negative regulation of Irf4 expression and accumulation of IRF4+ cells in the tissue and, consequently, suppresses the post-ischemic kidney immune response. Thus, we identified MCPIP1 as an important molecular sentinel of immune homeostasis in experimental acute kidney injury (AKI) and renal fibrosis.

Hippo-YAP/MCP-1 mediated tubular maladaptive repair promote inflammation in renal failed recovery after ischemic AKI.

Acute kidney injury (AKI) is associated with significant morbidity and its chronic inflammation contributes to subsequent chronic kidney disease (CKD) development. Yes-associated protein (YAP), the major transcriptional coactivator of the Hippo pathway, has been shown associated with chronic inflammation, but its role and mechanism in AKI-CKD transition remain unclear. Here we aimed to investigate the role of YAP in AKI-induced chronic inflammation. Renal ischemia/reperfusion (I/R) was used to induce a mouse model of AKI-CKD transition. We used verteporfin (VP), a pharmacological inhibitor of YAP, to treat post-IRI mice for a period, and evaluated the influence of YAP inhibition on long-term outcomes of AKI. In our results, severe IRI led to maladaptive tubular repair, macrophages infiltration, and progressive fibrosis. Following AKI, the Hippo pathway was found significantly altered with YAP persistent activation. Besides, tubular YAP activation was associated with the maladaptive repair, also correlated with interstitial macrophage infiltration. Monocyte chemoattractant protein 1 (MCP-1) was found notably upregulated with YAP activation. Of note, pharmacological inhibition of YAP in vivo attenuated renal inflammation, including macrophage infiltration and MCP-1 overexpression. Consistently, in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) induced YAP activation and MCP-1 overproduction whereas these could be inhibited by VP. In addition, we modulated YAP activity by RNA interference, which further confirmed YAP activation enhances MCP-1 expression. Together, we concluded tubular YAP activation with maladaptive repair exacerbates renal inflammation probably via promoting MCP-1 production, which contributes to AKI-CKD transition.

The CCL2/CCL7/CCL12/CCR2 pathway is substantially and persistently upregulated in mice after traumatic brain injury, and CCL2 modulates the complement system in microglia.

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1ß mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.

Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, but its complex pathogenesis is only insufficiently understood, resulting in still limited treatment options. OBJECTIVE: We sought to characterize AD on both transcriptomic and proteomic levels in humans. METHODS: We used skin suction blistering, a painless and nonscarring procedure that can simultaneously sample skin cells and interstitial fluid. We then compared results with conventional biopsies. RESULTS: Suction blistering captured epidermal and most immune cells equally well as biopsies, except for mast cells and nonmigratory CD163+ macrophages that were only present in biopsy isolates. Using single-cell RNA sequencing, we found comparable transcriptional profiles of key inflammatory pathways between blister and biopsy AD, but suction blistering was superior in cell-specific resolution for high-abundance transcripts (KRT1/KRT10, KRT16/KRT6A, S100A8/S100A9), which showed some background signals in biopsy isolates. Compared with healthy controls, we found characteristic upregulation of AD-typical cytokines such as IL13 and IL22 in Th2 and Th22 cells, respectively, but we also discovered these mediators in proliferating T cells and natural killer T cells, that also expressed the antimicrobial cytokine IL26. Overall, not T cells, but myeloid cells were most strongly enriched in AD, and we found dendritic cell (CLEC7A, amphiregulin/AREG, EREG) and macrophage products (CCL13) among the top upregulated proteins in AD blister fluid proteomic analyses. CONCLUSION: These data show that by using cutting-edge technology, suction blistering offers several advantages over conventional biopsies, including better transcriptomic resolution of skin cells, combined with proteomic information from interstitial fluid, unraveling novel inflammatory players that shape the cellular and proteomic microenvironment of AD.

Diverse Injury Pathways Induce Alveolar Epithelial Cell CCL2/12, Which Promotes Lung Fibrosis.

Accumulating evidence suggests that fibrosis is a multicellular process with contributions from alveolar epithelial cells (AECs), recruited monocytes/macrophages, and fibroblasts. We have previously shown that AEC injury is sufficient to induce fibrosis, but the precise mechanism remains unclear. Several cell types, including AECs, can produce CCL2 and CCL12, which can promote fibrosis through CCR2 activation. CCR2 signaling is critical for the initiation and progression of pulmonary fibrosis, in part through recruitment of profibrotic bone marrow-derived monocytes. Attempts at inhibiting CCL2 in patients with fibrosis demonstrated a marked upregulation of CCL2 production and no therapeutic response. To better understand the mechanisms involved in CCL2/CCR2 signaling, we generated mice with conditional deletion of CCL12, a murine homolog of human CCL2. Surprisingly, we found that mice with complete deletion of CCL12 had markedly increased concentrations of other CCR2 ligands and were not protected from fibrosis after bleomycin injury. In contrast, mice with lung epithelial cell-specific deletion of CCL12 were protected from bleomycin-induced fibrosis and had expression of CCL2 and CCL7 similar to that of control mice treated with bleomycin. Deletion of CCL12 within AECs led to decreased recruitment of exudate macrophages. Finally, injury to murine and human primary AECs resulted in increased production of CCL2 and CCL12, in part through activation of the mTOR pathway. In conclusion, these data suggest that targeting CCL2 may be a viable antifibrotic strategy once the pathways involved in the production and function of CCL2 and other CCR2 ligands are better defined.

Damaged male germ cells induce epididymitis in mice.

Epididymitis can be caused by infectious and noninfectious etiological factors. While microbial infections are responsible for infectious epididymitis, the etiological factors contributing to noninfectious epididymitis remain to be defined. The present study demonstrated that damaged male germ cells (DMGCs) induce epididymitis in mice. Intraperitoneal injection of the alkylating agent busulfan damaged murine male germ cells. Epididymitis was observed in mice 4 weeks after the injection of busulfan and was characterized by massive macrophage infiltration. Epididymitis was coincident with an accumulation of DMGCs in the epididymis. In contrast, busulfan injection into mice lacking male germ cells did not induce epididymitis. DMGCs induced innate immune responses in epididymal epithelial cells (EECs), thereby upregulating the pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), as well as the chemokines such as monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-5 (MCP-5), and chemokine ligand-10 (CXCL10). These results suggest that male germ cell damage may induce noninfectious epididymitis through the induction of innate immune responses in EECs. These findings provide novel insights into the mechanisms underlying noninfectious epididymitis, which might aid in the diagnosis and treatment of the disease.

Profiles of Local and Systemic Inflammation in the Outcome of Treatment of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia).

The immune mechanisms that contribute to the efficacy of treatment of cutaneous leishmaniasis (CL) are not fully understood. The aim of this study was to define immune correlates of the outcome of treatment of CL caused by Leishmania (Viannia) species during standard of care treatment with pentavalent antimonials. We conducted a comparative expression profiling of immune response genes in peripheral blood mononuclear cells (PBMCs) and lesion biopsy specimens obtained from CL patients before and at the end of treatment (EoT) with meglumine antimoniate. The ex vivo response of PBMCs to L (V) panamensis partially reflected that of lesion microenvironments. Significant downregulation of gene expression profiles consistent with local innate immune responses (monocyte and neutrophil activation and chemoattractant molecules) was observed at EoT in biopsy specimens of patients who cured (n = 8), compared to those from patients with treatment failure (n = 8). Among differentially expressed genes, pretreatment expression of CCL2 was significantly predictive of the therapeutic response (receiver operating characteristic [ROC] curve, area under the curve [AUC] = 0.82, P = 0.02). Polymorphisms in regulatory regions of the CCL2 promoter were analyzed in a pilot cohort of DNA samples from CL patients (cures, n = 20, and treatment failure, n = 20), showing putative association of polymorphisms rs13900(C/T) and rs2857656(G/C) with treatment outcome. Our data indicate that dampening gene expression profiles of monocyte and neutrophil activation characterize clinical cure after treatment of CL, supporting participation of parasite-sustained inflammation or deregulated innate immune responses in treatment failure.

A dual inhibitor of the proteasome catalytic subunits LMP2 and Y attenuates disease progression in mouse models of Alzheimer's disease.

The immunoproteasome (iP) is a variant of the constitutive proteasome (cP) that is abundantly expressed in immune cells which can also be induced in somatic cells by cytokines such as TNF-α or IFN-γ. Accumulating evidence support that the iP is closely linked to multiple facets of inflammatory response, eventually leading to the development of several iP inhibitors as potential therapeutic agents for autoimmune diseases. Recent studies also found that the iP is upregulated in reactive glial cells surrounding amyloid ß (Aß) deposits in brains of Alzheimer's disease (AD) patients, but the role it plays in the pathogenesis of AD remains unclear. In this study, we investigated the effects of several proteasome inhibitors on cognitive function in AD mouse models and found that YU102, a dual inhibitor of the iP catalytic subunit LMP2 and the cP catalytic subunit Y, ameliorates cognitive impairments in AD mouse models without affecting Aß deposition. The data obtained from our investigation revealed that YU102 suppresses the secretion of inflammatory cytokines from microglial cells. Overall, this study indicates that there may exist a potential link between LMP2/Y and microglia-mediated neuroinflammation and that inhibition of these subunits may offer a new therapeutic strategy for AD.

Synchrotron Microbeam Radiation Therapy as a New Approach for the Treatment of Radioresistant Melanoma: Potential Underlying Mechanisms.

PURPOSE: Synchrotron microbeam radiation therapy (MRT) is a method that spatially distributes the x-ray beam into several microbeams of very high dose (peak dose), regularly separated by low-dose intervals (valley dose). MRT selectively spares normal tissues, relative to conventional (uniform broad beam [BB]) radiation therapy. METHODS AND MATERIALS: To evaluate the effect of MRT on radioresistant melanoma, B16-F10 murine melanomas were implanted into mice ears. Tumors were either treated with MRT (407.6 Gy peak; 6.2 Gy valley dose) or uniform BB irradiation (6.2 Gy). RESULTS: MRT induced significantly longer tumor regrowth delay than did BB irradiation. A significant 24% reduction in blood vessel perfusion was observed 5 days after MRT, and the cell proliferation index was significantly lower in melanomas treated by MRT compared with BB. MRT provoked a greater induction of senescence in melanoma cells. Bio-Plex analyses revealed enhanced concentration of monocyte-attracting chemokines in the MRT group: MCP-1 at D5, MIP-1α, MIP-1ß, IL12p40, and RANTES at D9. This was associated with leukocytic infiltration at D9 after MRT, attributed mainly to CD8 T cells, natural killer cells, and macrophages. CONCLUSIONS: In light of its potential to disrupt blood vessels that promote infiltration of the tumor by immune cells and its induction of senescence, MRT could be a new therapeutic approach for radioresistant melanoma.

Anatomical structure, and expression of CCL4 and CCL13-like during the development of maxillary barbel in Paramisgurnus dabryanus.

Paramisgurnus dabryanus is one of the most economically important fishes in China. Barbels are an essential sensory organ for the food-seeking ability of teleost fish. However, the anatomical structure of the maxillary barbels of P. dabryanus and the molecular basis of their development are unknown. We investigated the anatomical structure of the barbel, and gene expression patterns of two chemokine C-C motif ligands: CCL4 and CCL13-like during the maxillary barbel development using Masson Trichrome staining, light and electron microscopy, and qPCR. Anatomically, the maxillary barbel of P. dabryanus contains taste buds, melanophores, collagen fibers, connective tissue, smooth muscles, nerve bundles, and blood vessels, but does not have skeletal muscles or a skeleton rod. The expression of CCL4 and CCL13-like was weak or non-existent in the early phases of development, but high at the last two studied time-points: 192- and 216-h post-hatching. Results indicated that CCL4 and CCL13-like were related to the development of the maxillary barbel.

Treatment with andrographolide sulfonate provides additional benefits to imipenem in a mouse model of Klebsiella pneumoniae pneumonia.

Klebsiella pneumoniae is a primary cause of community-acquired and nosocomial respiratory infections, and K. pneumoniae resistance to the current treatment approach with carbapenem is worsening. Andrographolide is a natural diterpenoid from Andrographis paniculata that was shown to exert anti-inflammatory activity. We herein show that pretreatment with a water-soluble andrographolide sulfonate significantly attenuate lung injury and infiltration of inflammatory cells. Interestingly, mice receiving combined treatment with andrographolide sulfonate displayed perfect survival rate than the mice treatment with imipenem alone, and monocyte chemotactic protein 5 (MCP-5) level was decreased further. These findings suggest that andrographolide sulfonate could as a potential synergist for antibiotic treatment of bacteria-induced inflammation.

Chemokines CCL2 and CCL7, but not CCL12, play a significant role in the development of pain-related behavior and opioid-induced analgesia.

The complex neuroimmunological interactions mediated by chemokines are suggested to be responsible for the development of neuropathic pain. The lack of knowledge regarding the detailed pathomechanism of neuropathy is one reason for the lack of optimally efficient therapies. Recently, several lines of evidence indicated that expression of CCR2 is increased in spinal cord neurons and microglial cells after peripheral nerve injury. It was previously shown that administration of CCR2 antagonists induces analgesic effects; however, the role of CCR2 ligands in neuropathic pain still needs to be explained. Thus, the goal of our studies was to investigate the roles of CCL2, CCL7, and CCL12 in neuropathic pain development and opioid effectiveness. The experiments were conducted on primary glial cell cultures and two groups of mice: naive and neuropathic. We used chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model. Mice intrathecally received chemokines (CCL2, CCL7, CCL12) at a dose of 10, 100 or 500 ng, neutralizing antibodies (anti-CCL2, anti-CCL7) at a dose of 1, 4 or 8 µg, and opioids (morphine, buprenorphine) at a dose of 1 µg. The pain-related behaviors were assessed using the von Frey and cold plate tests. The biochemical analysis of mRNA expression of glial markers, CCL2, CCL7 and CCL12 was performed using quantitative reverse transcriptase real-time PCR. We demonstrated that CCI of the sciatic nerve elevated spinal expression of CCL2, CCL7 and CCL12 in mice, in parallel with microglia and astroglial activation markers. Moreover, intrathecal injection of CCL2 and CCL7 induced pain-related behavior in naive mice in a dose-dependent manner. Surprisingly, intrathecal injection of CCL12 did not influence nociceptive transmission in naive or neuropathic mice. Additionally, we showed for the first time that intrathecal injection of CCL2 and CCL7 neutralizing antibodies not only attenuated CCI-induced pain-related behaviors in mice but also augmented the analgesia induced by morphine and buprenorphine. In vitro studies suggest that both microglia and astrocytes are an important cellular sources of the examined chemokines. Our results revealed the crucial roles of CCL2 and CCL7, but not CCL12, in neuropathic pain development and indicated that pharmacological modulation of these factors may serve as a potential therapeutic target for new (co)analgesics.

STAT3 inhibition reduces macrophage number and tumor growth in neurofibroma.

Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK2/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition downregulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, Iba1+; F4/80+;CD11b+ macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed macrophage proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation, and decreased Schwann cell proliferation and increased Schwann cell death. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.

The CC chemokines CCL8, CCL13 and CCL20 are local inflammatory biomarkers of HLA-B27-associated uveitis.

PURPOSE: To determine the concentrations of the CC chemokines CCL2, CCL7, CCL8, CCL11, CCL13, CCL20, CCL24 and CCL26 in aqueous humour (AH) samples from patients with specific uveitic entities. METHODS: Aqueous humour samples from patients with active uveitis associated with Behçet's disease (BD) (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12) and control patients (n = 9) were assayed with the use of a multiplex assay. RESULTS: When considering all uveitis patients as one group, all chemokine levels except CCL2 were significantly increased compared to controls. CCL8, CCL13 and CCL20 were the most strongly upregulated, 48-fold, 118-fold and 173-fold, respectively, above control AH levels. CCL8 and CCL13 levels were significantly higher in HLA-B27-associated uveitis than in sarcoidosis and VKH disease. CCL20 levels were significantly higher in HLA-B27-associated uveitis than in BD, sarcoidosis and VKH disease. In addition, CCL20 levels were significantly higher in BD than in VKH disease. In HLA-B27-associated uveitis, CCL8, CCL13 and CCL20 were upregulated 111-fold, 255-fold and 465-fold, respectively, compared with controls. CCL8, CCL13 and CCL20 levels were significantly higher in nongranulomatous uveitis (BD and HLA-B27-associated uveitis) than in granulomatous uveitis (sarcoidosis and VKH disease). CONCLUSION: Immune responses mediated by CCL8, CCL13 and CCL20 appear to be more potent in nongranulomatous uveitis, particularly in HLA-B27-associated uveitis.

Borrelia burgdorferi basic membrane protein A stimulates murine macrophage to secrete specific chemokines.

In this study, we investigated the mechanisms that lead to the production of proinflammatory mediators by the murine macrophage cell line, RAW264.7, when these cells are exposed in vitro to recombinant Borrelia burgdorferi basic membrane protein A (rBmpA). Using antibody protein microarray technology with high-throughput detection ability for detecting 25 chemokines in culture supernatant the RAW264.7 cell culture supernatants at 12 and 24 h post-stimulation with rBmpA, we identified two chemokines, a monocyte chemoattractant protein-5 (MCP-5/CCL12) and a macrophage inflammatory protein-2 (MIP-2/CXCL2), both of which increased significantly after stimulation. We then chose these two chemokines for further study. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction revealed that with the increase of rBmpA concentration, MCP-5/CCL12 and MIP-2/CXCL2 showed concentration-dependent increases (p <0.01).Our results indicate that the rBmpA could stimulate the secretion of several specific chemokines and induce Lyme arthritis.

B-cell leukemia/lymphoma 10 promotes angiogenesis in an experimental corneal neovascularization model.

PURPOSE: Corneal neovascularization (CrNV) arises from many causes including corneal inflammatory, infectious, or traumatic insult, and frequently leads to impaired vision. This study seeks to determine the role of B-cell leukemia/lymphoma 10 (BCL-10) in the development of experimental CrNV. METHODS: Corneas from BCL-10 knockout (KO) mice and wild-type (WT) mice were burned by sodium hydroxide (NaOH) to create the CrNV model and neovascular formation in the corneas was assessed 2 weeks later. Intracorneal macrophage accumulation and the expression of angiogenic factors were quantified by flow cytometric analysis (FCM) and real-time PCR, respectively. RESULTS: The amount of CrNV was determined 2 weeks after alkali burn. Compared to WT mice, the amount of CrNV in BCL-10 KO mice was significantly decreased. FCM revealed that F4/80-positive macrophages were markedly decreased in BCL-10 KO mice compared with WT mice. Reverse transcription PCR showed that the mRNA expression levels of intracorneal vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF) and monocyte chemotactic protein 1 were reduced in BCL-10 KO mice compared with WT mice. CONCLUSION: BCL-10 KO mice exhibited reduced alkali-induced CrNV by suppressing intracorneal macrophage infiltration, which subsequently led to decreased VEGF-A and bFGF expression, suggesting that BCL-10 may become a potential clinical intervening target of CrNV.

Periodontal-induced chronic inflammation triggers macrophage secretion of Ccl12 to inhibit fibroblast-mediated cardiac wound healing.

Chronic inflammatory diseases, such as periodontal disease, associate with adverse wound healing in response to myocardial infarction (MI). The goal of this study was to elucidate the molecular basis for impaired cardiac wound healing in the setting of periodontal-induced chronic inflammation. Causal network analysis of 168 inflammatory and extracellular matrix genes revealed that chronic inflammation induced by a subseptic dose of Porphyromonas gingivalis lipopolysaccharide (LPS) exacerbated infarct expression of the proinflammatory cytokine Ccl12. Ccl12 prevented initiation of the reparative response by prolonging inflammation and inhibiting fibroblast conversion to myofibroblasts, resulting in diminished scar formation. Macrophage secretion of Ccl12 directly impaired fibronectin and collagen deposition and indirectly stimulated collagen degradation through upregulation of matrix metalloproteinase-2. In post-MI patients, circulating LPS levels strongly associated with the Ccl12 homologue monocyte chemotactic protein 1 (MCP-1). Patients with LPS levels ≥ 1 endotoxin units (EU)/ml (subseptic endotoxemia) at the time of hospitalization had increased end diastolic and systolic dimensions compared with post-MI patients with < 1 EU/ml, indicating that low yet pathological concentrations of circulating LPS adversely impact post-MI left ventricle (LV) remodeling by increasing MCP-1. Our study provides the first evidence to our knowledge that chronic inflammation inhibits reparative fibroblast activation and generates an unfavorable cardiac-healing environment through Ccl12-dependent mechanisms.