Ingrid Stroo

Chemokine expression in renal ischemia/reperfusion injury is most profound during the reparative phase

Chemokines are important players in the migration of leukocytes to sites of injury and are also involved in angiogenesis, development and wound healing. In this study, we performed microarray analyses to identify chemokines that play a role during the inflammatory and repair phase after renal ischemia/reperfusion (I/R) injury and investigated the temporal relationship between chemokine expression, leukocyte accumulation and renal damage/repair. C57Bl/6 mice were subjected to unilateral ischemia for 45 min and sacrificed 3 h, 1 day and 7 days after reperfusion. From ischemic and contralateral kidney, RNA was isolated and hybridized to a microarray. Microarray results were validated with quantitative real-time reverse transcription–PCR (QRT–PCR) on RNA from an independent experiment. (Immuno)histochemical analyses were performed to determine renal damage/repair and influx of leukocytes. Twenty out of 114 genes were up-regulated at one or more reperfusion periods. All these genes were up-regulated 7 days after I/R. Up-regulated genes included CC chemokines MCP-1 and TARC, CXC chemokines KC and MIP-2α, chemokine receptors Ccr1 and Cx3cr1 and related genes like matrix metalloproteinases. Microarray data of 1 and 7 days were confirmed for 17 up-regulated genes by QRT–PCR. (Immuno)histochemical analysis showed that the inflammatory and repair phase after renal I/R injury take place after, respectively, 1 and 7 days. Interestingly, chemokine expression was highest during the repair phase. In addition, expression profiles showed a biphasic expression of all up-regulated CXC chemokines coinciding with the early inflammatory and late repair phase. In conclusion, we propose that temporal expression of chemokines is a crucial factor in the regulation of renal I/R injury and repair.

Lipopolysaccharide Inhibits Th2 Lung Inflammation Induced by House Dust Mite Allergens in Mice

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 μg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.

Haematopoietic stem cell migration to the ischemic damaged kidney is not altered by manipulating the SDF-1/CXCR4-axis

Background. Haematopoietic stem cells (HSC) have been shown to migrate to the ischemic kidney. The factors that regulate the trafficking of HSC to the ischemic damaged kidney are not fully understood. The stromal cell-derived factor-1 (SDF-1)/CXCR4-axis has been identified as the central signalling axis regulating trafficking of HSC to the bone marrow. Therefore, we hypothesized that SDF-1/CXCR4 interactions are implicated in the migration of HSC to the injured kidney. Methods. HSC were isolated from mouse bone marrow and labelled with a cell tracker. Acceptor mice were subjected to unilateral ischemia and received HSC intravenously directly after reperfusion. In addition, in separate groups of acceptor mice, endogenous SDF-1 or HSC-associated CXCR4 was blocked or kidneys were injected with SDF-1. Results. Exogenous HSC could be detected in the tubules and interstitium of the kidney 24 h after ischemic injury. Importantly, the amount of HSC in the ischemic kidney was markedly higher compared to the contralateral kidney. Neutralizing endogenous SDF-1 or HSC-associated CXCR4 did not prevent the migration of HSC. No increase in the number of labelled HSC could be observed after local administration of SDF-1, as was also determined in bilateral kidney ischemia. Conclusion. In conclusion, systemically administered HSC preferentially migrate to the ischemic injured kidney. This migration could not be prevented by blocking the SDF-1/CXCR4-axis or increased after local administration of SDF-1.

Protease-activated receptor-2 deficient mice have reduced house dust mite-evoked allergic lung inflammation

Protease-activated receptor-2 (PAR2) is abundantly expressed in the pulmonary compartment. House dust mite (HDM) is a common cause of allergic asthma and contains multiple PAR2 agonistic proteases. The aim of this study was to determine the role of PAR2 in HDM-induced allergic lung inflammation. For this, the extent of allergic lung inflammation was studied in wild type (Wt) and PAR2 knockout (KO) mice after repeated airway exposure to HDM. HDM exposure of Wt mice resulted in a profound influx of eosinophils in bronchoalveolar lavage fluid (BALF) and accumulation of eosinophils in lung tissue, which both were strongly reduced in PAR2 KO mice. PAR2 KO mice demonstrated attenuated lung pathology and protein leak in the bronchoalveolar space, accompanied by lower BALF levels of the anaphylatoxins C3a and C5a. This study reveals, for the first time, an important role for PAR2 in allergic lung inflammation induced by the clinically relevant allergens contained in HDM.

Stem Cell Factor Expression after Renal Ischemia Promotes Tubular Epithelial Survival

Background Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF) has been shown to protect the tubular epithelium against apoptosis. Methodology/Principal Findings In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. Conclusions/Significance Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

The Toll Interleukin-1 Receptor (IL-1R) 8/Single Ig Domain IL-1R-Related Molecule Modulates the Renal Response to Bacterial Infection

ABSTRACT Our immune system has to constantly strike a balance between activation and inhibition of an inflammatory response to combat invading pathogens and avoid inflammation-induced collateral tissue damage. Toll interleukin-1 receptor 8 (IL-1R-8)/single Ig domain IL-1R-related molecule (TIR8/SIGIRR) is an inhibitor of Toll-like receptor (TLR)/IL-1R signaling, which is predominantly expressed in the kidney. The biological role of renal TIR8 during infection is, however, unknown. We therefore evaluated renal TIR8 expression during Escherichia coli pyelonephritis and explored its role in host defense using TIR8−/− versus TIR8+/+ mice. We found that TIR8 protein is abundantly present in the majority of cortical tubular epithelial cells. Pyelonephritis resulted in a significant downregulation of TIR8 mRNA in kidneys of TIR8+/+ mice. TIR8 inhibited an effective host response against E. coli, as indicated by diminished renal bacterial outgrowth and dysfunction in TIR8−/− mice. This correlated with increased amounts of circulating and intrarenal neutrophils at the early phase of infection. TIR8−/− tubular epithelial cells had increased cytokine/chemokine production when stimulated with lipopolysaccharide (LPS) or heat-killed E. coli, suggesting that TIR8 played an anti-inflammatory role during pathogen stimulation by inhibiting LPS signaling. These data suggest that TIR8 is an important negative regulator of an LPS-mediated inflammatory response in tubular epithelial cells and dampens an effective antibacterial host response during pyelonephritis caused by uropathogenic E. coli.

Depletion of Gut Microbiota Protects against Renal Ischemia-Reperfusion Injury

An accumulating body of evidence shows that gut microbiota fulfill an important role in health and disease by modulating local and systemic immunity. The importance of the microbiome in the development of kidney disease, however, is largely unknown. To study this concept, we depleted gut microbiota with broad-spectrum antibiotics and performed renal ischemia-reperfusion (I/R) injury in mice. Depletion of the microbiota significantly attenuated renal damage, dysfunction, and remote organ injury and maintained tubular integrity after renal I/R injury. Gut flora-depleted mice expressed lower levels of F4/80 and chemokine receptors CX3CR1 and CCR2 in the F4/80+ renal resident macrophage population and bone marrow (BM) monocytes than did control mice. Additionally, compared with control BM monocytes, BM monocytes from gut flora-depleted mice had decreased migratory capacity toward CX3CL1 and CCL2 ligands. To study whether these effects were driven by depletion of the microbiota, we performed fecal transplants in antibiotic-treated mice and found that transplant of fecal material from an untreated mouse abolished the protective effect of microbiota depletion upon renal I/R injury. In conclusion, we show that depletion of gut microbiota profoundly protects against renal I/R injury by reducing maturation status of F4/80+ renal resident macrophages and BM monocytes. Therefore, dampening the inflammatory response by targeting microbiota-derived mediators might be a promising therapy against I/R injury.

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation

Bacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.

Deficiency for the chemokine monocyte chemoattractant protein-1 aggravates tubular damage after renal ischemia/reperfusion injury.

Temporal expression of chemokines is a crucial factor in the regulation of renal ischemia/reperfusion (I/R) injury and repair. Beside their role in the migration and activation of inflammatory cells to sites of injury, chemokines are also involved in other processes such as angiogenesis, development and migration of stem cells. In the present study we investigated the role of the chemokine MCP-1 (monocyte chemoattractant protein-1 or CCL2), the main chemoattractant for monocytes, during renal I/R injury. MCP-1 expression peaks several days after inducing renal I/R injury coinciding with macrophage accumulation. However, MCP-1 deficient mice had a significant decreased survival and increased renal damage within the first two days, i.e. the acute inflammatory response, after renal I/R injury with no evidence of altered macrophage accumulation. Kidneys and primary tubular epithelial cells from MCP-1 deficient mice showed increased apoptosis after ischemia. Taken together, MCP-1 protects the kidney during the acute inflammatory response following renal I/R injury.

Myeloid-related protein-14 deficiency promotes inflammation in staphylococcal pneumonia

Staphylococcus aureus has evolved as an important cause of pneumonia in both hospital and community settings. Staphylococcal lung infection can lead to overwhelming pulmonary inflammation. During infection, neutrophils release complexes of myeloid-related protein (MRP)8 and MRP14 (MRP8/14). MRP8/14 has been shown to exert pro-inflammatory and chemotactic activity, and to assist in the killing of S. aureus. In the current study we sought to determine the role of MRP8/14 in the host response during S. aureus pneumonia. Pneumonia was induced in wildtype and MRP14-deficient mice (mice unable to form MRP8/14) by intranasal inoculation of 1×107 CFU of S. aureus USA300. Mice were sacrificed at 6, 24, 48 or 72 h after infection for analyses. S. aureus pneumonia was associated with a strong rise in MRP8/14 in bronchoalveolar lavage fluid and lung tissue. Surprisingly, MRP14 deficiency had a limited effect on bacterial clearance and was associated with increased cytokine levels in bronchoalveolar lavage fluid and aggravated lung histopathology. MRP14 deficiency in addition was associated with a diminished transmigration of neutrophils into bronchoalveolar lavage fluid at late time-points after infection together with reduced release of nucleosomes. MRP8/14 serves in an unexpected protective role for the lung in staphylococcal pneumonia. MRP8/14 unexpectedly protects against excessive pulmonary inflammation in murine staphylococcal pneumonia http://ow.ly/IwYt6