CSF-1R inhibition attenuates ischemia-induced renal injury and fibrosis by reducing Ly6C+ M2-like macrophage infiltration.

Acute kidney injury (AKI) to chronic kidney disease (CKD) progression has become a life-threatening disease. However, an effective therapeuticstrategyis still needed. The pathophysiology of AKI-to-CKD progression involves chronic inflammation and renal fibrosis driven by macrophage activation, which is physiologically dependent on colony-stimulating factor-1 receptor (CSF-1R) signaling. In this study, we modulated macrophage infiltration through oral administration of the CSF-1R inhibitor GW2580 in an ischemia-reperfusion (I/R)-induced AKI model to evaluate its therapeutic effects on preventing the progression of AKI to CKD. We found that GW2580 induced a significant reduction in the number of macrophages in I/R-injured kidneys and attenuated I/R-induced renal injury and subsequent interstitial fibrosis. By flow cytometry, we observed that the reduced macrophages were primarily Ly6C+ inflammatory macrophages in the GW2580-treated kidneys, while there was no significant difference in the number and percentage of Ly6C-CX3CR1+ macrophages. We further found that these reduced macrophages also demonstrated some characteristics of M2-like macrophages, which have been generally regarded as profibrotic subtypes in chronic inflammation. These results indicate the existence of phenotypic and functional crossover between Ly6C+ and M2-like macrophages in I/R kidneys, which induces AKI worsening to CKD. In conclusion, therapeutic GW2580 treatment alleviates acute renal injury and subsequent fibrosis by reducing Ly6C+ M2-like macrophage infiltration in ischemia-induced AKI.

Novel cholinergic peptides SLURP-1 and -2 regulate epithelialization of cutaneous and oral wounds.

It is well established that auto/paracrine acetylcholine (ACh) is essential for wound epithelialization, and that the mechanisms include regulation of keratinocyte motility and adhesion via nicotinic ACh receptors (nAChRs). Keratinocyte nAChRs can be also activated by non-canonical ligands, such as secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and -2. In this study, we determined effects of recombinant (r)SLURP-1 and-2 on migration of human epidermal and oral keratinocytes under agarose and epithelialization of cutaneous and oral mucosal excisional wounds in mice, and also identified nAChRs mediating SLURP signals. Both in vitro and in vivo, rSLURP-1 decreased and SLURP-2 increased epithelialization rate. The mixture of both peptides accelerated epithelialization even further, indicating that their simultaneous signaling renders an additive physiologic response. The specificity of rSLURP actions was illustrated by similar effects on cutaneous and oral wounds, which feature distinct responses to injury, and also by abrogation of rSLURP effects with neutralizing antibodies. rSLURP-1 acted predominantly via the α7 nAChR-coupled up-regulation of the sedentary integrins α2 and α3 , whereas SLURP-2--through α3, and α9 nAChRs up-regulating migratory integrins α5 and αV . The biologic effects of rSLURPs required the presence of endogenous ACh, indicating that auto/paracrine SLURPs provide for a fine tuning of the physiologic regulation of crawling locomotion via the keratinocyte ACh axis. Since nAChRs have been shown to regulate SLURP production, cholinergic regulation of keratinocyte migration appears to be mediated by a reciprocally arranged network. The cholinergic peptides, therefore, may become prototype drugs for the treatment of wounds that fail to heal.

Role of fibulin-5 in metastatic organ colonization.

The tumor microenvironment is now recognized as a major factor in determining the survival and growth of disseminated tumor cells at potential metastatic sites. Tumor cells send signals to stroma cells and stimulate them to produce factors that in turn create favorable conditions for tumor cell metastasis. Activated fibroblasts constitute an important component of the tumor-associated stroma. We have previously shown that S100A4 protein produced by stromal fibroblasts in the primary tumor stimulates metastasis formation. Here we show that activated fibroblasts also stimulate the formation of metastases independently of S100A4 expression during organ colonization. To identify genes that could potentially interfere with fibroblast-driven metastasis, we used gene expression profiling of S100A4-deficient fibroblasts treated with and without tumor cell-conditioned media. Five differentially expressed genes encoding cell surface and secreted proteins with potential metastasis-modulating activity were selected. Expression of lymphocyte antigen 6 complex (Ly6c) and matrix metalloproteinase 3 (Mmp3) was upregulated in fibroblasts in response to tumor-conditioned medium, whereas expression of cadherin-16 (Cdh16), Ccn2, and fibulin-5 (Fbln5) was downregulated. Further analysis showed that Fibulin-5 is able to suppress the metastatic colonization of lungs and liver. Additional studies suggest a mechanism in which Fibulin-5 suppresses metastasis formation by inhibiting production of matrix metalloproteinase 9 (MMP9) and reducing the invasive behavior of fibroblasts. Together our data are consistent with the notion that tumors secrete factors that downregulate expression of Fbln5 in fibroblasts at sites of metastatic colonization, in turn upregulating Mmp9 expression and stimulating metastatic organ colonization.

Cementoblast gene expression is regulated by Porphyromonas gingivalis lipopolysaccharide partially via toll-like receptor-4/MD-2.

Lipopolysaccharides are potent inflammatory mediators considered to contribute to destruction of periodontal tissues. Here, we hypothesized that Porphyromonas gingivalis lipopolysaccharide (P-LPS) treatment would regulate gene expression in murine cementoblasts through Toll-like receptor 4. Real-time (RT)-PCR and Northern blot analysis indicated that P-LPS decreased expression of transcripts for osteocalcin (OCN) and receptor activator of nuclear factor kappaB ligand (RANKL). In contrast, a dose-dependent up-regulation in mRNA levels for osteopontin (OPN) and osteoprotegerin (OPG) was observed. Similarly, ELISA demonstrated decreased RANKL and increased OPG levels. A monoclonal antibody specific for mouse TLR-4/MD-2 partially neutralized the P-LPS effect on cementoblasts. These results indicate that exposure of cementoblasts to P-LPS can alter cell function by regulating markers of osteoclastic activity (e.g., RANKL/OPG), thereby potentially affecting the inflammation-associated resorption of mineralized tissues.

Augmentation of NK cell-mediated cytotoxicity to tumor cells by inhibitory NK cell receptor blockers.

NK cells monitor expression of MHC class I by inhibitory receptors and preferentially kill cells that lose or down-regulate MHC class I expression. One possible mechanism by which tumor cells evade NK cell killing is continued expression of appropriate MHC class I ligands to engage inhibitory receptors on NK cells. We show here that small-mol.-wt blockers against the mouse inhibitory NK cell receptor Ly49A enhance NK cell killing of such tumor cells. We identified Ly49A-binding peptides by selecting phages with the capacity to bind recombinant Ly49A expressed in Escherichia coli from a phage display random peptide library. The Ly49A-binding peptides could also bind Ly49A expressed on mammalian cells. Importantly, the Ly49A-binding peptides blocked Ly49A recognition of its MHC class I ligands H-2Dd and H-2Dk. Moreover, blockade of Ly49A by the peptides enhanced cytotoxicity of Ly49A+ NK cells towards H-2Dd-expressing tumor cells. These results clearly indicate effectiveness of small-mol.-wt blockers of inhibitory NK cell receptors in enhancing NK cell-mediated killing of tumor cells that are otherwise resistant because of MHC class I expression.

NK inhibitory-receptor blockade for purging of leukemia: effects on hematopoietic reconstitution.

One of the obstacles of BMT that limits its efficacy is failure to eradicate the original tumor. The incidence of tumor relapse is particularly high after autologous BMT. Natural killer (NK) cells comprise various subsets that can express inhibitory receptors for MHC class I determinants. We have recently demonstrated that blockade of NK-cell inhibitory receptors can augment antitumor effects in vitro and in vivo. However, breakdown of tolerance and autoreactivity may occur as a result of the inhibition of NK-cell inactivation to self MHC determinants. We have utilized F(ab')2 fragments of monoclonal antibody, 5E6, against Ly49C/I inhibitory receptors, which are expressed on 35% to 60% of NK cells in H2b strains of mice and are specific for H2Kb, to investigate the effect of inhibitory-receptor blockade on syngeneic bone marrow cell (BMC) and tumor cell growth. We show that treatment of interleukin 2-activated C57BL/6 (B6, H2b) SCID-mouse NK cells with 5E6 F(ab')2 fragments during 48-hour coculture resulted in autoreactivity against syngeneic BMCs as demonstrated by suppression of myeloid reconstitution on day 14 post-BMT. However, this suppressive effect was transient and normalized by day 21 post-BMT. In contrast, blockade of inhibitory receptors during 24-hour coculture had no adverse effects on myeloid reconstitution after BMT. Furthermore, under the same coculture conditions, NK cell-mediated purging of C1498 leukemia cells contaminating syngeneic BMCs was more effective with inhibitory-receptor blockade, leading to a significantly higher proportion of animals with long-term survival compared to the control recipients. These results demonstrate that short-term in vitro blockade of inhibitory receptors can augment antitumor activity without long-term inhibitory effects on BMCs and thus may be of potential use in the purging of contaminating tumor cells prior to autologous BMT.

Natural killer cell proliferation induced by anti-NK1.1 and IL-2.

NKR-P1 molecules are involved in natural killing of certain tumour targets. Indeed, the NK1.1 (NKR-P1C) molecule is the most specific serological marker on murine NK cells in C57BL/6 mice. Previous studies of NKR-P1 have indicated that anti-NKR-P1 mAb induced NK cells to kill otherwise insensitive targets, NK cell phosphoinositol turnover and Ca++ flux but it was not previously known if all NK cells were activated. In this study we report that immobilized anti-NK1.1 also specifically induced proliferation as measured by thymidine incorporation. The response required low doses of IL-2 for a synergistic effect. Cells stimulated with anti-NK1.1 + IL-2 displayed characteristic cytolytic activity against a NK-sensitive tumour target, YAC-1. However, anti-NK1.1-stimulated cells displayed delayed proliferation kinetics, heterogeneity of the expression of the very early antigen marker, CD69, and altered expression of the Ly-49 family members when compared to NK cells activated by high concentrations of IL-2. Taken together, these data demonstrate that immobilized anti-NK1.1 triggers only a subpopulation of NK cells.