Collectin 11 has a pivotal role in host defense against kidney and bladder infection in mice.

The urinary tract is constantly exposed to microorganisms. Host defense mechanisms in protection from microbial colonization and development of urinary tract infections require better understanding to control kidney infection. Here we report that the lectin collectin 11 (CL-11), particularly kidney produced, has a pivotal role in host defense against uropathogen infection. CL-11 was found in mouse urine under normal and pathological conditions. Mice with global gene ablation of Colec11 had increased susceptibility to and severity of kidney and to an extent, bladder infection. Mice with kidney-specific Colec11 ablation exhibited a similar disease phenotype to that observed in global Colec11 deficient mice, indicating the importance of kidney produced CL-11 for protection against kidney and bladder infection. Conversely, intravesical or systemic administration of recombinant CL-11 reduced susceptibility to and severity of kidney and bladder infection. Mechanism analysis revealed that CL-11 can mediate several key innate defense mechanisms (agglutination, anti- adhesion, opsonophagocytosis), and limit local inflammatory responses to pathogens. Furthermore, CL-11-mediated innate defense mechanisms can act on clinically relevant microorganisms including multiple antibiotic resistant strains. CL-11 was detectable in eight of 24 urine samples from patients with urinary tract infections but not detectable in urine samples from ten healthy individuals. Thus, our findings demonstrate that CL-11 is a key factor of host defense mechanisms in kidney and bladder infection with therapeutic potential for human application.

Protective role for C3aR in experimental chronic pyelonephritis.

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.

Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.

The C5a/C5aR1 axis promotes progression of renal tubulointerstitial fibrosis in a mouse model of renal ischemia/reperfusion injury.

C5a is a potent proinflammatory agonist that mediates renal ischemia reperfusion (IR) injury, but the potential for modulating chronic post-ischemic fibrosis and use of therapeutic antagonist are undefined. Here we determine whether C5a receptor 1 (C5aR1) signaling is essential to the development of post-ischemic fibrosis and if it is a valid target for therapeutic blockade with soluble receptor antagonist. C5aR1 is required for the development of renal tubulointerstitial fibrosis in a murine model of renal ischemia/reperfusion injury. Deficiency of C5aR1 protected mice from the development of the fibrosis. This protection was associated with attenuated deposition of extracellular matrix components (fibronectin, collagen I), reduced cellular infiltrates (CD45, F4/80), and gene expression of proinflammatory and profibrogenic mediators in the kidney. In an in vitro model of hypoxia/reoxygenation, C5a stimulation caused renal fibroblast proliferation and activation, and upregulated gene expression of interleukin-1α (IL-1α), IL-6 and transforming growth factor-α (TGF-α) in renal tubular epithelial cells and monocytes/macrophages. Administration of a C5aR1 antagonist (PMX53) significantly reduced renal injury and tubulointerstitial fibrosis. Thus, our results demonstrate a pathogenic role for C5aR1 in the progression of tubulointerstitial fibrosis following renal IR injury and support that C5aR1-mediated local inflammatory responses to hypoxic renal injury contribute to tubulointerstitial fibrosis through several cellular pathways, namely, promoting tubule injury, interstitial fibroblast proliferation and epithelial-to-mesenchymal transition of renal tubular epithelial cells. Our results also suggest the C5a-C5aR1 interaction is a therapeutic target for chronic post-ischemic fibrosis.

The C3a/C3aR axis mediates anti-inflammatory activity and protects against uropathogenic E coli-induced kidney injury in mice.

Both the C3a/C3aR and C5a/C5aR1 axes are regarded as important pathways for inducing and regulating inflammatory responses. It is well documented that the C5a/C5aR1 axis is a potent inflammatory mediator in the pathogenesis of many clinic disorders. However, our understanding of the role of the C3a/C3aR axis in renal disorders remains limited. Contrary to the C5a/C5aR axis, we now show that the C3a/C3aR axis has a protective role in uropathogenic Escherichia coli (UPEC)-induced renal injury. C3aR-/- mice were found to develop severe renal pathology compared to wild type mice, a pathology characterized by intense tissue damage and an increased bacterial load within the kidney. This was associated with an overwhelming production of pro-inflammatory mediators and increased neutrophil infiltration in the kidney. Bone marrow chimera experiments found that tissue damage and bacterial load were significantly reduced in C3aR-/- mice that received bone marrow from wild type mice, compared with that in mice re-populated with bone marrow from C3aR-/- mice. This supports a critical role for C3aR on myeloid cells in the pathological process. Pharmacological treatment of mice with a C3aR agonist reduced both the extent of tissue injury and bacterial load. Mechanistic analyses indicated that the C3a/C3aR axis downregulates the lipopolysaccharide-induced pro-inflammatory responses in macrophages and facilitates the phagocytosis of UPEC by phagocytes. Thus, our findings clearly demonstrate a protective role of the C3a/C3aR axis in UPEC-induced renal injury, conferred by the suppression of pro-inflammatory responses and enhanced phagocytosis by macrophages.

The complement factor 5a receptor 1 has a pathogenic role in chronic inflammation and renal fibrosis in a murine model of chronic pyelonephritis.

Complement factor 5a (C5a) interaction with its receptor (C5aR1) contributes to the pathogenesis of inflammatory diseases, including acute kidney injury. However, its role in chronic inflammation, particularly in pathogen-associated disorders, is largely unknown. Here we tested whether the development of chronic inflammation and renal fibrosis is dependent on C5aR1 in a murine model of chronic pyelonephritis. C5aR1-deficient (C5aR1-/-) mice showed a significant reduction in bacterial load, tubule injury and tubulointerstitial fibrosis in the kidneys following infection, compared with C5aR1-sufficient mice. This was associated with reduced renal leukocyte infiltration specifically for the population of Ly6Chi proinflammatory monocytes/macrophages and reduced intrarenal gene expression of key proinflammatory and profibrogenic factors in C5aR1-/- mice following infection. Antagonizing C5aR1 decreased renal bacterial load, tissue inflammation and tubulointerstitial fibrosis. Ex vivo and in vitro studies showed that under infection conditions, C5a/C5aR1 interaction upregulated the production of proinflammatory and profibrogenic factors by renal tubular epithelial cells and monocytes/macrophages, whereas the phagocytic function of monocytes/macrophages was down-regulated. Thus, C5aR1-dependent bacterial colonization of the tubular epithelium, C5a/C5aR1-mediated upregulation of local inflammatory responses to uropathogenic E. coli and impairment of phagocytic function of phagocytes contribute to persistent bacterial colonization of the kidney, chronic renal inflammation and subsequent tubulointerstitial fibrosis.

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 °C, and gently suspended in culture medium using a 200 µL pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached >90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Collectin-11 promotes cancer cell proliferation and tumor growth.

Collectin-11 (CL-11) is a recently described soluble C-type lectin that has distinct roles in embryonic development, host defence, autoimmunity, and fibrosis. Here we report that CL-11 also plays an important role in cancer cell proliferation and tumor growth. Melanoma growth was found to be suppressed in Colec11-/- mice in a s.c. B16 melanoma model. Cellular and molecular analyses revealed that CL-11 is essential for melanoma cell proliferation, angiogenesis, establishment of more immunosuppressive tumor microenvironment, and the reprogramming of macrophages to M2 phenotype within melanomas. In vitro analysis revealed that CL-11 can activate tyrosine kinase receptors (EGFR, HER3) and ERK, JNK, and AKT signaling pathways and has a direct stimulatory effect on murine melanoma cell proliferation. Furthermore, blockade of CL-11 (treatment with L-fucose) inhibited melanoma growth in mice. Analysis of open data sets revealed that COLEC11 gene expression is upregulated in human melanomas and that high COLEC11 expression has a trend toward poor survival. CL-11 also had direct stimulatory effects on human tumor cell proliferation in melanoma and several other types of cancer cells in vitro. Overall, our findings provide the first evidence to our knowledge that CL-11 is a key tumor growth-promoting protein and a promising therapeutic target in tumor growth.

The C5a/C5aR1 Axis Contributes to the Pathogenesis of Acute Cystitis Through Enhancement of Adhesion and Colonization of Uropathogenic E. coli.

Our previous work using a murine model of pyelonephritis demonstrated that the C5a/C5aR1 axis plays a pathogenic role in acute kidney infection. In this study, we report that the C5a/C5aR1 axis also plays a pathogenic role in acute bladder infection. C5aR1-deficient mice had reduced bladder bacterial load and attenuated bladder tissue injury, which is associated with reduced expression of terminal α-mannosyl residues (Man) (a potential ligand for type 1 fimbriae of E. coli) at the luminal surface of the bladder epithelium and reduced early bacterial colonization of the bladder. In vitro, C5a stimulation enhanced mannose expression in and facilitated bacterial adhesion/colonization to human bladder epithelial cells. C5a stimulation also upregulated the activation of ERK1/2 and NF-κB signaling and gene expression of proinflammatory cytokines (i.e., Il6, Il1b, Cxcl1, Ccl2) in the epithelial cells, which could drive pro-inflammatory responses leading to tissue injury. Administration of the C5aR1 antagonist effectively reduced bladder bacterial load and tissue injury. Thus, our findings demonstrate a previously unknown pathogenic role for the C5a/C5aR1 axis in bladder infection and suggest that the C5a/C5aR1 axis-mediated upregulation of Man expression, enhancement of bacterial adhesion/colonization, and excessive inflammatory responses contribute to acute bladder infection. These findings improve our understanding of the pathogenesis of bladder infection with therapeutic implications for UTI.

The C5a/C5aR2 axis promotes renal inflammation and tissue damage.

C5a is a potent inflammatory mediator that binds C5aR1 and C5aR2. Although pathogenic roles of the C5a/C5aR1 axis in inflammatory disorders are well documented, the roles for the C5a/C5aR2 axis in inflammatory disorders and underlying mechanisms remain unclear. Here, we show that the C5a/C5aR2 axis contributes to renal inflammation and tissue damage in a mouse model of acute pyelonephritis. Compared with WT littermates, C5ar2-/- mice had significantly reduced renal inflammation, tubular damage, and renal bacterial load following bladder inoculation with uropathogenic E. coli. The decrease in inflammatory responses in the kidney of C5ar2-/- mice was correlated with reduced intrarenal levels of high mobility group box-1 protein (HMGB1), NLRP3 inflammasome components, cleaved caspase-1, and IL-1ß. In vitro, C5a stimulation of macrophages from C5ar1-/- mice (lacking C5aR1 but expressing C5aR2) led to significant upregulation of HMGB1 release, NLRP3/cleaved caspase-1 inflammasome activation, and IL-1ß secretion. Furthermore, blockade of HMGB1 significantly reduced C5a-mediated upregulation of NLRP3/cleaved caspase-1 inflammasome activation and IL-1ß secretion in the macrophages, implying a HMGB1-dependent upregulation of NLRP3/cleaved caspase-1 inflammasome activation in macrophages. Our findings demonstrate a pathogenic role for the C5a/C5aR2 axis in renal injury following renal infection and suggest that the C5a/C5aR2 axis contributes to renal inflammation and tissue damage through upregulation of HMGB1 and NLRP3/cleaved caspase-1 inflammasome.

Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells.

Recent work in a murine model of ascending urinary tract infection has suggested that C5a/C5aR1 interactions play a pathogenic role in the development of renal infection through enhancement of bacterial adhesion/colonization to renal tubular epithelial cells (RTECs). In the present study, we extended these observations to human. We show that renal tubular epithelial C5aR1 signaling is involved in promoting uropathogenic Escherichia coli (UPEC) adhesion/invasion of host cells. Stimulation of primary cultures of RTEC with C5a resulted in significant increases in UPEC adhesion/invasion of the RTEC. This was associated with enhanced expression of terminal α-mannosyl residues (Man) (a ligand for type 1 fimbriae of E. coli) in the RTEC following C5a stimulation. Mechanism studies revealed that C5aR1-mediated activation of ERK1/2/NF-κB and upregulation of proinflammatory cytokine production (i.e., TNF-α) is at least partly responsible for the upregulation of Man expression and bacterial adhesion. Clinical sample studies showed that C5aR1 and Man were clearly detected in the renal tubular epithelium of normal human kidney biopsies, and UPEC bound to the epithelium in a d-mannose-dependent manner. Additionally, C5a levels were significantly increased in urine of urinary tract infection patients compared with healthy controls. Our data therefore demonstrate that, in agreement with observations in mice, human renal tubular epithelial C5aR1 signaling can upregulate Man expression in RTEC, which enhances UPEC adhesion to and invasion of RTEC. It also suggests the in vivo relevance of upregulation of Man expression in renal tubular epithelium by C5a/C5aR1 interactions and its potential impact on renal infection.

C5aR1 promotes acute pyelonephritis induced by uropathogenic E. coli.

C5a receptor 1 (C5aR1) is a G protein-coupled receptor for C5a and also an N-linked glycosylated protein. In addition to myeloid cells, C5aR1 is expressed on epithelial cells. In this study, we examined the role of C5aR1 in bacterial adhesion/colonization of renal tubular epithelium and addressed the underlying mechanisms of this role. We show that acute kidney infection was significantly reduced in mice with genetic deletion or through pharmacologic inhibition of C5aR1 following bladder inoculation with uropathogenic E. coli (UPEC). This was associated with reduced expression of terminal α-mannosyl residues (Man; a ligand for type 1 fimbriae of E. coli) on the luminal surface of renal tubular epithelium and reduction of early UPEC colonization in these mice. Confocal microscopy demonstrated that UPEC bind to Man on the luminal surface of renal tubular epithelium. In vitro analyses showed that C5a stimulation enhances Man expression in renal tubular epithelial cells and subsequent bacterial adhesion, which, at least in part, is dependent on TNF-α driven by C5aR1-mediated intracellular signaling. Our findings demonstrate a previously unknown pathogenic role for C5aR1 in acute pyelonephritis, proposing a potentially novel mechanism by which C5a/C5aR1 signaling mediates upregulation of carbohydrate ligands on renal tubules to facilitate UPEC adhesion.