Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi.

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2(-/-) mice harbored up to 100-fold more spirochetes in tissues than did TLR2(+/+) littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2(-/-) mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2(-/-) mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2(-/-) mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.     

MyD88 plays a unique role in host defense but not arthritis development in Lyme disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88(-/-) mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88(-/-) mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88(-/-) mice responded poorly to Borrelia sonicate in vitro, MyD88(-/-) mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.     

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.     

Toll-like receptor 3 is an essential component of the innate stress response in virus-induced cardiac injury

Enterovirus-induced myocardial injury can lead to severe heart failure. To date, little is known about the early innate stress response that contributes to host defense in the heart. Toll-like receptor 3 (TLR3) is important in the initiation of the innate antiviral response. We investigated the involvement of TLR3, which recognizes viral double-stranded RNA, on encephalomyocarditis virus (EMCV) infection. To examine the contribution of TLR3 in protection from EMCV infection, we infected mice deficient in TLR3 with 50 plaque-forming units of EMCV. TLR3-deficient (TLR3(-/-)) mice were more susceptible to EMCV infection and had a significantly higher viral load in the heart compared with TLR3(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TLR3(-/-) than in TLR3(+/+)mice. TLR3(-/-) mice had impaired proinflammatory cytokine and chemokine expression in the heart following EMCV infection. However, the expression of interferon-beta was not impaired in EMCV-infected TLR3(-/-) mice. EMCV infection leads to a TLR3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury.     

The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.     

Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.     

Genetic basis of murine antibacterial defense to streptococcal lung infection

To evaluate the effect of genetic background on antibacterial defense to streptococcal infection, eight genetically diverse strains of mice (A/J, DBA/2J, CAST/Ei, FVB/NJ, BALB/cJ, C57BL/6J, 129/SvImJ, and C3H/HeJ) and tlr2-deficient mice (C57BL/6^tlr2−/−) were infected with three doses of Streptococcus zooepidemicus (500, 5,000, or 50,000 colony-forming units) by alveolar challenge. There was a range of susceptibility between the strains at each dose and time point (6, 24, and 96 h). At the lowest dose, the 129/SvImJ and C3H/HeJ strains had significantly higher bacterial counts at all time points after infection, when compared to A/J, DBA/2J, CAST/Ei, FVB/NJ, which were resistant to infection at the low dose of innoculum. At the medium dose, 129/SvImJ and C3H/HeJ had higher bacterial counts, while A/J, DBA/2J, and BALB/cJ showed reduced streptococcal growth. After the highest dose of Streptococcus, there were minimal differences between strains, suggesting the protective impact of modifier genes can be overcome. TLR2-deficient animals contained increased bacterial load with reduced cytokines after 96 h when compared to C57BL/6J controls suggesting a role of innate immunity in late antibacterial defense. Overall, we identify vulnerable (129/SvlmJ and C3H/HeJ) and resistant (A/J, FVB, and DBA) mouse strains to streptococcal lung infection, which demonstrate divergent genetic expression profiles. These results demonstrate that innate differences in pulmonary host defense to S. zooepidemicus are dependent on host genetic factors.     

Gene expression profiling reveals unique pathways associated with differential severity of lyme arthritis

The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligonucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 mice, because C57BL/6-IL-10(-/-) mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-kappaB-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis.     

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.     

Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-kappaB signaling pathway and up-regulation of cytokine synthesis such as TNF-alpha and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.     

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.     

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-alpha and IL-6. Administration of anti-TNF-alpha Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-alpha as the molecular mediator of bacterial clearance and septic shock.     

Deficiency in TLR4 signal transduction ameliorates cardiac injury and cardiomyocyte contractile dysfunction during ischemia

Toll-like receptor 4 (TLR4), a proximal signalling receptor in innate immune responses to lipopolysaccharide of gram-negative pathogens, is expressed in the heart. Accumulating evidence have consolidated the notion that TLR4 plays an essential role in the pathogenesis of cardiac dysfunction. However, the molecular mechanisms of TLR4 responsible for ischemia-induced cardiac dysfunction remain unclear. To address the signalling mechanisms of TLR4-deficiency cardioprotection against ischemic injury, in vivo regional ischemia was induced by occlusion of the left anterior descending coronary artery in wild-type (WT) C3H/HeN and TLR4-mutated C3H/HeJ mice. The results demonstrated that blunted ischemic activation of p38 mitogen-activated protein kinase and JNK signalling occurred in C3H/HeJ hearts versus C3H/HeN hearts, while ERK and AMP-activated protein kinase (AMPK) signalling pathways were augmented during ischemia in C3H/HeJ hearts versus C3H/HeN hearts. Intriguingly, ischemia-stimulated endoplasmic reticulum stress was higher in C3H/HeN hearts than that in C3H/HeJ as demonstrated by up-regulation of Grp78/BiP, Gadd153/CHOP and IRE-1α. Myocardial infarct, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining demonstrated that C3H/HeN hearts suffered more damage than those of C3H/HeJ hearts during ischemia. Moreover, isolated cardiomyocytes from C3H/HeJ hearts showed resistance to hypoxia-induced contractile dysfunction compared to those from C3H/HeN hearts, which are associated with greater hypoxic activation of AMPK and ERK signalling, better intracellular Ca²⁺ handling in C3H/HeJ versus C3H/HeN cardiomyocytes. These findings suggest that the cardioprotective effects against ischemic injury of hearts with deficiency in TLR4 signalling may be mediated through modulating AMPK and ERK signalling pathway during ischemia.     

Control of Mycobacterium bovis BCG infection with increased inflammation in TLR4-deficient mice

Live mycobacteria have been reported to signal through several pattern recognition receptors (PRR), among them toll-like receptor 4 (TLR4) and TLR2 in vitro. Here, we investigated the role of TLR4 in host resistance to Mycobacterium bovis (BCG) infection in vivo. In vitro, macrophages of TLR4 mutant C3H/HeJ mice infected with BCG expressed lower levels of TNF than controls, and TNF release was further decreased, although not completely absent, in the absence of TLR2. In vivo, TLR4 mutant C3H/HeJ and control C3H/HeOUJ mice were infected with BCG (2 x 10(6) CFU i.v.). Both TLR4 mutant and wild-type mice were able to control the infection and survived 8 months post-BCG infection. Macrophage activation with abundant acid-fast bacilli and expression of inducible nitric oxide synthase (iNOS) and MHC class II antigens was seen in both groups of mice. However, TLR4 mutant mice experienced an arrest of body weight gain and showed signs of increased inflammation, with persistent splenomegaly, increase in granuloma number and augmented neutrophil infiltration. Infection of TLR4-deficient mice with higher doses of BCG (1 and 3 x 10(7) CFU, i.v.) increased the inflammation in spleen and liver, associated with a transient, higher bacterial load in the liver. In summary, TLR4 mutant mice show normal macrophage recruitment and activation, granuloma formation and control of the BCG infection, but this is associated with persistent inflammation. Therefore, TLR4 signaling is not essential for early control of BCG infection, but it may have a critical function in fine tuning of inflammation during chronic mycobacterial infection.     

Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.