Epithelial G protein-coupled receptor kinases regulate the initial inflammatory response during mycobacterial infection.

The interaction between mycobacteria and epithelium is unexplored, but may determine the outcome of the infection. We have analyzed the role of two G protein-coupled receptors, CXCR1 and CXCR2 that are important regulators of many pulmonary diseases. We found that mycobacteria significantly increased the expression of both CXCR1 and CXCR2 on alveolar epithelial cells and both receptors were found to be important for neutrophil diapedesis across primary endothelial cells towards infected mucosa. Mycobacteria, lipoarabinomannan or 19-kDa glycolipoprotein up-regulated the inhibitory G protein-coupled receptor kinase (GRK)2, while GRK3 was less affected. Mycobacteria-induced GRK2 up-regulation decreased chemokine transcription and secretion thereby affecting the neutrophil recruitment to infected mucosa. These events were completely abolished by blocking these receptors prior to infection as the blocking increased epithelial immune responses. We have identified novel interactions occurring in the initial phase of mycobacterial infections by which mycobacterial manipulate epithelial inflammatory responses.

Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses.

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence.

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice.

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2(+/-)) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2(-/-)) and wild-type (mCxcr2(+/+)) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis.

HAMLET treatment delays bladder cancer development.

PURPOSE: HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. MATERIALS AND METHODS: Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. RESULTS: HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. CONCLUSIONS: Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development.

Therapeutic effect of blocking CXCR2 on neutrophil recruitment and dextran sodium sulfate-induced colitis.

Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 anti-serum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis.

Shiga toxin-mediated disease in MyD88-deficient mice infected with Escherichia coli O157:H7.

Toll-like receptors (TLRs) are key factors of innate immunity that detect pathogen invasion and trigger a host response. TLR4 can mediate a response through adaptor molecules, MyD88 or TRIF. In the present study, streptomycin-treated MyD88(-/-), Tlr4(-/-), Trif (Lps2/Lps2), and C57BL/6 wild-type (WT) mice were infected with either Shiga toxin (Stx)-producing or non-producing Escherichia coli O157:H7. Moderate to severe clinical signs of disease developed in MyD88(-/-) (n = 21/21), Tlr4(-/-) (n = 12/16), Trif (Lps2/Lps2) (n = 7/15) and WT mice (n = 6/20) infected with Stx-producing E. coli O157:H7 but not in mice inoculated with the Stx non-producing strain (n = 0/54, P < 0.001). MyD88(-/-) mice infected with Stx-producing E. coli O157:H7 developed the most severe disease and had the highest bacterial burden. Hematological analysis of sick MyD88(-/-) mice showed reduced red blood cell counts and reticulocytosis, suggesting hemolysis. Thrombocytopenia developed in MyD88(-/-), Trif (Lps2/Lps2), and WT mice, and creatinine levels were elevated in both MyD88(-/-) and WT mice infected with the Stx-producing strain. Renal histopathology showed evidence of glomerular capillary congestion, tubular desquamation, and fibrinogen deposition, and intestinal histopathology showed mucosal injury, edema, and inflammation in sick mice. Administration of purified Stx2 to MyD88(-/-) and WT mice led to severe disease in both groups, suggesting that MyD88(-/-) mice are not more sensitive to Stx than WT mice. As MyD88(-/-) mice developed the most severe disease hematological and pathological changes, the results suggest that dysfunctional innate immune responses via MyD88 enhanced Stx-induced disease.

Glycolipid receptor depletion as an approach to specific antimicrobial therapy.

Mucosal pathogens recognize glycoconjugate receptors at the site of infection, and attachment is an essential first step in disease pathogenesis. Inhibition of attachment may prevent disease, and several approaches have been explored. This review discusses the prevention of bacterial attachment and disease by agents that modify the glycosylation of cell surface glycoconjugates. Glycosylation inhibitors were tested in the urinary tract infection model, where P-fimbriated Escherichia coli rely on glycosphingolipid receptors for attachment and tissue attack. N-butyldeoxynojirimycin blocked the expression of glucosylceramide-derived glycosphingolipids and attachment was reduced. Bacterial persistence in the kidneys was impaired and the inflammatory response was abrogated. N-butyldeoxynojirimycin was inactive against strains which failed to engage these receptors, including type 1 fimbriated or nonadhesive strains. In vivo attachment has been successfully prevented by soluble receptor analogues, but there is little clinical experience of such inhibitors. Large-scale synthesis of complex carbohydrates, which could be used as attachment inhibitors, remains a technical challenge. Antibodies to bacterial lectins involved in attachment may be efficient inhibitors, and fimbrial vaccines have been developed. Glycosylation inhibitors have been shown to be safe and efficient in patients with lipid storage disease and might therefore be tested in urinary tract infection. This approach differs from current therapies, including antibiotics, in that it targets the pathogens which recognize these receptors.

Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.

This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHEC-LPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected on platelets from 12 of 14 children with EHEC-associated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n = 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS.

Uropathogenic Escherichia coli as a model of host-parasite interaction.

Resistance to mucosal infection varies greatly in the population, but the molecular basis of disease susceptibility is often unknown. Studies of host-pathogen infections are helpful to identify virulence factors, which characterise disease isolates, and successful defence strategies of hosts that resist infection. In the urinary tract infection (UTI) model, we have identified crucial steps in the pathogen-activated innate host response, and studied the genetic control of these activation steps. Furthermore, genetic variation in the innate host-response defence is investigated as a basis of disease susceptibility. The Toll-like receptor 4 (TLR4) controls initial mucosal response to uropathogenic Escherichia coli (UPEC). Bacterial TLR4 activation in epithelial cells leads to chemokine secretion and neutrophil recruitment and TLR4 mutant mice develop an asymptomatic carrier state. The chemokine receptor CXCR1 determines the efficiency of neutrophil migration and activation, and thus of bacterial clearance. CXCR1 mutant mice become bacteremic and develop renal scars and studies in UTI prone children have detected low CXCR1 expression, suggesting that CXCR1 is also essential for human disease susceptibility.

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4.

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6-16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.

Natural history of renal scarring in susceptible mIL-8Rh-/- mice.

BACKGROUND: Urinary tract infections (UTIs) cause end-stage renal disease (ESRD) but the molecular mechanisms have remained unclear. Recently, the interleukin (IL)-8 receptor was shown to control disease susceptibility in mice and low IL-8 receptor expression was observed in pyelonephritis-prone patients. METHODS: Intravesical Escherichia coli infection was established in mIL-8Rh-/- or Balb/c control mice. Survival, bacterial persistence, and histology were used as measurements of disease severity. RESULTS: Within 2 days, 19/30 mIL-8Rh-/- mice developed lethal infection with bacteremia. Surviving mice remained infected and developed progressive renal damage with pathologic neutrophil accumulation and abscess formation first under the pelvic epithelium and then throughout the tissue. Recruited immune effector cells were unable to remove the dying neutrophils and frustrated macrophages formed foam cell aggregates. As a result, there was successive destruction of the mucosal barrier, medulla and cortex and necrosis of the renal papilla. The mIL-8Rh+/+ mice all survived and infection was cleared within a few days without symptoms or tissue pathology. CONCLUSION: mIL-8Rh-/- mice develop acute bacteremic pyelonephritis and renal scarring due to a dysfunctional neutrophil response. The tissue damage resembles human disease, and these mice offer a model system to study the molecular mechanisms of renal scarring.

Glycolipid depletion in antimicrobial therapy.

Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N-butyldeoxynojirimycin (NB-DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P-fimbriated Escherichia coli. NB-DNJ blocks the ceramide-specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose-dependent way, and depletion markedly inhibited P-fimbriated bacterial attachment in vitro. NB-DNJ-fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P-fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae-mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.