TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.     

Enhanced airway reactivity and inflammation in A2A adenosine receptor-deficient allergic mice

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.     

IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice

Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors (>95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of PGE(2), a well-known inhibitor of gel contraction, was higher (P < 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1beta and tumor necrosis factor-alpha, whereas fibroblasts increased synthesis of PGE(2). Antibodies to interleukin-1beta and tumor necrosis factor-alpha blocked the monocyte inhibitory effect and reduced the amount of PGE(2) produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling.     

Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively. A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type 2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble ST2 directly bound to IL-33 and suppressed activation of NF-kappaB in EL-4 cells stably expressing ST2L, suggesting that the complex of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates the biological activity of IL-33 in allergic airway inflammation.     

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.     

Sulfuretin attenuates allergic airway inflammation in mice

Sulfuretin is one of the main flavonoids produced by Rhus verniciflua, which is reported to inhibit the inflammatory response by suppressing the NF-κB pathway. Because NF-κB activation plays a pivotal role in the pathogenesis of allergic airway inflammation, we here examined the effect of sulfuretin on an ovalbumin-induced airway inflammation model in mice. We isolated sulfuretin from R. verniciflua. Sulfuretin was delivered intraperitoneally after the last ovalbumin challenge. Airway hyper-responsiveness, cytokines, mucin, and eosinophilic infiltration were analyzed in bronchoalveolar lavage fluid and lung tissue. A single administration of sulfuretin reduced airway inflammatory cell recruitment and peribronchiolar inflammation and suppressed the production of various cytokines in bronchoalveolar fluid. In addition, sulfuretin suppressed mucin production and prevented the development of airway hyper-responsiveness. The protective effect of sulfuretin was mediated by the inhibition of the NF-κB signaling pathway. Our results suggest that sulfuretin may have therapeutic potential for the treatment of allergic airway inflammation.     

TLR2-mediated production of IL-27 and chemokines by respiratory epithelial cells promotes bleomycin-induced pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis is a fatal disease characterized by progressive destruction of the lung. Although TLR2 bridges innate and adaptive immunity by sensing tissue damage, its role in pulmonary fibrosis remains unclear. To address this issue, TLR2(-/-) and WT mice were examined for bleomycin-induced pulmonary fibrosis (BIPF). Flow cytometric and immunohistochemical analysis revealed that TLR2 expression in bronchial epithelial and immune cells of the lungs was upregulated in WT mice during BIPF. Levels of IL-27, TGF-β, chemokines, and hydroxyproline were lower in lungs of TLR2(-/-) mice than in those of WT mice, but IL-17 levels were higher in TLR2(-/-) mice. In in vivo experiments using bone marrow-chimeric mice, TLR2 expression on respiratory epithelial cells, rather than immune cells, induced IL-27 and chemokine production in the lungs, further stimulating BIPF. This effect of TLR2 depended on IRF complexes and MyD88. BIPF was more severe in IL-17A(-/-) mice and in TLR2(-/-) mice treated with anti-IL-17 mAb than in TLR2(-/-) and WT mice. Furthermore, IL-27 blockade in WT mice reduced hydroxyproline levels by enhancing IL-17 production, whereas the treatment of TLR2(-/-) mice with a chemokine mixture increased hydroxyproline levels by recruiting inflammatory cells into the lungs. TLR2 signaling promotes BIPF by inducing IL-27 and chemokine production by respiratory epithelial cells, thereby inhibiting IL-17 production and recruiting inflammatory cells into the lungs.     

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.     

A3 adenosine receptor signaling contributes to airway inflammation and mucus production in adenosine deaminase-deficient mice

Adenosine signaling has been implicated in chronic lung diseases such as asthma and chronic obstructive pulmonary disease; however, the specific roles of the various adenosine receptors in processes central to these disorders are not well understood. In this study, we have investigated the role(s) of the A(3) adenosine receptor in adenosine-dependent pulmonary inflammation observed in adenosine deaminase (ADA)-deficient mice. The A(3) receptor (A(3)R) was found to be expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A(3)R antagonist, prevented airway eosinophilia and mucus production. Similar findings were seen in the lungs of ADA/A(3) double knockout mice. Although eosinophils were decreased in the airways of ADA-deficient mice following antagonism or removal of the A(3)R, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A(3)R is needed for the migration of eosinophils into the airways. These findings identify an important role for the A(3)R in regulating lung eosinophilia and mucus production in an environment of elevated adenosine.     

Mycoplasma pneumoniae infection increases airway collagen deposition in a murine model of allergic airway inflammation

Mycoplasma pneumoniae (Mp) has been linked to chronic asthma. Airway remodeling (e.g., airway collagen deposition or fibrosis) is one of the pathological features of chronic asthma. However, the effects of respiratory Mp infection on airway fibrosis in asthma remain unclear. In the present study, we hypothesized that respiratory Mp infection may increase the airway collagen deposition in a murine model of allergic airway inflammation in part through upregulation of transforming growth factor (TGF)-beta1. Double (2 wk apart) inoculations of Mp or saline (control) were given to mice with or without previous allergen (ovalbumin) challenges. On days 14 and 42 after the last Mp or saline, lung tissue and bronchoalveolar lavage (BAL) fluid were collected for analyses of collagen and TGF-beta1 at protein and mRNA levels. In allergen-na?ve mice, Mp did not alter airway wall collagen. In allergen-challenged mice, Mp infections did not change airway wall collagen deposition on day 14 but increased the airway collagen on day 42; this increase was accompanied by increased TGF-beta1 protein in the airway wall and reduced TGF-beta1 protein release from the lung tissue into BAL fluid. Our results suggest that Mp infections could modulate airway collagen deposition in a murine model of allergic airway inflammation with TGF-beta1 involved in the collagen deposition process.     

Reduction of allergic airway responses in P-selectin-deficient mice

De Sanctis, George T., Walter W. Wolyniec, Francis H. Y. Green, Shixin Qin, Aiping Jiao, Patricia W. Finn, Thomas Noonan, Anthony A. Joetham, Erwin Gelfand, Claire M. Doerschuk, and Jeffrey M. Drazen. Reduction of allergic airway responses inP-selectin-deficient mice. J. Appl.Physiol. 83(3): 681-687, 1997.P-selectin is an adhesion receptor that has been shown to be important in therecruitment of eosinophils and lymphocytes in a variety of inflammatoryconditions. Because cellular recruitment is thought to be a criticalevent in allergen-induced changes in airway responsiveness, we reasoned that P-selectin-deficient mice would exhibit reduced airwayresponsiveness and cellular trafficking noted in wild-type (+/+) mice.Both (+/+) and P-selectin-deficient (/) micesensitized and challenged with ovalbumin (OVA/OVA) exhibited thesame capacity to produce increased titers of total and OVA-specificimmunoglobulin E. Airway responsiveness to methacholine wassignificantly greater in the (+/+) (OVA/OVA) animals than it was in therespective (/) (OVA/OVA) group or control groups(P = 0.0016). Bronchoalveolarlavage fluid from (/) (OVA/OVA) mice contained significantlyfewer eosinophils and lymphocytes compared with the (+/+) (OVA/OVA)mice (P < 0.05). These resultssuggest that the predominant role of P-selectin in OVA-inducedairway hyperresponsiveness is to promote the airway inflammatoryresponse to allergen inhalation.

     

Modulation of allergic inflammation in mice deficient in TNF receptors

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.     

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10^-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10^-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.     

Melatonin prevents allergic airway inflammation in epicutaneously sensitized mice

Purpose: The pathological process of atopic dermatitis (AD) progressing into other types of allergic diseases such as asthma and allergic rhinitis during the first several years of life is often referred to as the atopic march. Although the phenomenon of atopic march has been recognized for decades, how asthma stems from AD is still not fully understood, confounding a universal strategy to effectively protect people from the atopic march. Methods: We established experimental atopic march mice by first inducing allergic dermatitis with 0.5% fluorescein isothiocyante (FITC) applied to the skin, followed by an ovalbumin (OVA) airway challenge. In addition, by examining serum immunoglobulin (Ig) concentrations, airway cytokines, the levels of oxidative stress markers, histopathological changes in lung tissue and airway hyperresponsiveness (AHR), we were able to validate the successful establishment of the model. Furthermore, by detecting the attenuating effects of melatonin (MT) and the levels of oxidative stress in the atopic march mice, we explored the potential molecular mechanisms involved in the development of atopic march. Results: By successfully establishing an experimental atopic march mouse model, we were able to demonstrate that overproduction of oxidative stress in the lung significantly up-regulated the activation of nuclear factor-κB (NF-κB) signaling pathways causing thymic stromal lymphopoietin (TSLP) release, which further promotes the development of atopic march. Conclusions: To mitigate the development of the atopic march, antioxidants such as MT may be imperative to inhibit NF-κB activation in the lung, especially after the onset of AD.     

A dipalmitoylated lipoprotein from Mycoplasma pneumoniae activates NF-kappa B through TLR1, TLR2, and TLR6

The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Recently, lipoproteins from mycoplasmas have been reported to induce NF-kappaB activation. In this study, we examined the ability of lipoproteins from M. pneumoniae to activate NF-kappaB, and the active component responsible for the NF-kappaB activation was identified. Lipid-associated membrane proteins from M. pneumoniae were found to induce NF-kappaB through TLR 2 in a human monocytic cell line, THP-1. The active component of the Lipid-associated membrane proteins was a subunit b of F0F1-type ATPase (F0F1-ATPase). The F0F1-ATPase is assumed to contain two palmitic acids. The activation of NF-kappaB by the F0F1-ATPase was inhibited by a dominant negative construct of TLR1 and TLR6. These results indicate that the activation of NF-kappaB by F0F1-ATPase is dependent on TLR1, TLR2, and TLR6. The activity of the F0F1-ATPase was decreased with pretreatment of lipoprotein lipase but not protease, indicating that the lipid moiety of the F0F1-ATPase was important for the NF-kappaB activation. Thus, a dipalmitoylated lipoprotein from M. pneumoniae was found to activate NF-kappaB through TLR1, TLR2, and TLR6.     

IL-4 promotes airway eosinophilia by suppressing IFN-gamma production: defining a novel role for IFN-gamma in the regulation of allergic airway inflammation

Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4(-/-) Th2 cells were transferred into IFN-gammaR(-/-) mice, indicating that IFN-gamma was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-gamma to mice after IL-4(+/+) Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-gamma. IFN-gamma reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-gamma, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.     

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.     

Fever range temperature promotes TLR4 expression and signaling in dendritic cells

Fever improves survival and shortens disease duration in microbial infections. However, the mechanisms of these beneficial responses still remain elusive. Toll-like receptors (TLRs) play important roles in sensing microbes invading and therefore we hypothesized that fever range temperature may enhance responsiveness of dendritic cells (DCs) to lipopolysaccharide (LPS) by promoting TLR4 expression and signaling. In this study, we found that pretreatment of DCs with 39.5 degrees C temperature can up-regulate TLR4 expression in DCs and enhances LPS-induced DC production of interleukins (IL) IL-6, IL-10 and IL-12 but not tumor necrosis factor alpha (TNF-alpha). Blockade of the autocrine action of IL-10 could increase LPS-induced TNF-alpha and IL-12 production in DCs. Further experiments confirmed that TLR4 ligation activates extracellular signal-regulated kinase (ERK), p38, and nuclear factor-kappaB pathways more potently in DCs pretreated with 39.5 degrees C. We conclude that fever range temperature can promote TLR4 expression and signaling in DCs, leading to enhancement of immune responses to inflammatory stimuli. These results might reveal a possible mechanistic explanation for the significance of fever in activating innate immune responses.     

Methotrexate-induced mucositis in mucin 2-deficient mice

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2(-/-)) and wild type (Muc2(+/+)) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2(+/+) and Muc2(-/-) mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2(+/+) and Muc2(-/-) mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2(+/+) mice showed a trend towards regaining weight, whereas Muc2(-/-) mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2(-/-) and Muc2(+/+) mice was comparable. Prior to MTX-injection, tumor necrosis factor-alpha and interleukin-10 mRNAs were upregulated in Muc2(-/-) mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment.