Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.     

TNF receptor signaling contributes to chemokine secretion, inflammation, and respiratory deficits during Pneumocystis pneumonia

CD8(+) T cells contribute to the pathophysiology of Pneumocystis pneumonia (PcP) in a murine model of AIDS-related disease. The present studies were undertaken to more precisely define the mechanisms by which these immune cells mediate the inflammatory response that leads to lung injury. Experimental mice were depleted of either CD4(+) T cells or both CD4(+) and CD8(+) T cells and then infected with Pneumocystis: The CD4(+)-depleted mice had significantly greater pulmonary TNF-alpha levels than mice depleted of both CD4(+) and CD8(+) T cells. Elevated TNF-alpha levels were associated with increased lung concentrations of the chemokines RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant. To determine whether TNFR signaling was involved in the CD8(+) T cell-dependent chemokine response, TNFRI- and II-deficient mice were CD4(+) depleted and infected with Pneumocystis: TNFR-deficient mice had significantly reduced pulmonary RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant responses, reduced inflammatory cell recruitment to the alveoli, and reduced histological evidence of PcP-related alveolitis as compared with infected wild-type mice. Diminished pulmonary inflammation correlated with improved surfactant activity and improved pulmonary function in the TNFR-deficient mice. These data indicate that TNFR signaling is required for maximal CD8(+) T cell-dependent pulmonary inflammation and lung injury during PcP and also demonstrate that CD8(+) T cells can use TNFR signaling pathways to respond to an extracellular fungal pathogen.     

Parenchymal cell TNF receptors contribute to inflammatory cell recruitment and respiratory failure in Pneumocystis carinii-induced pneumonia

The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4(+) T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8(+) T cells and the TNF receptors (TNFR) TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57BL6/J, and TNFRsf1a/1b double-null origin were generated, CD4(+) T cell depleted, and inoculated with Pc. As expected, C57 --> C57 chimeras (donor marrow --> recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance), and inflammatory cell infiltration. In contrast, TNFRsf1a/1b(-/-) --> TNFRsf1a/1b(-/-) mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57 --> TNFRsf1a/1b(-/-)) had milder disease than did C57 --> C57 mice. Both groups of mice with TNFR-deficient parenchymal cells had low bronchoalveolar lavage fluid total cell counts and fewer lavageable CD8(+) T cells than did C57 --> C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with wild-type parenchymal cells but TNFRsf1a/1b(-/-) hematopoietic cells (TNFRsf1a/1b(-/-) --> C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radiosensitive, bone marrow-derived cells.     

MyD88 is pivotal for the early inflammatory response and subsequent bacterial clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia

Chlamydia pneumoniae is the causative agent of respiratory tract infections and a number of chronic diseases. Here we investigated the involvement of the common TLR adaptor molecule MyD88 in host responses to C. pneumoniae-induced pneumonia in mice. MyD88-deficient mice were severely impaired in their ability to mount an acute early inflammatory response toward C. pneumoniae. Although the bacterial burden in the lungs was comparable 5 days after infection, MyD88-deficient mice exhibited only minor signs of pneumonia and reduced expression of inflammatory mediators. MyD88-deficient mice were unable to up-regulate proinflammatory cytokines and chemokines, demonstrated delayed recruitment of CD8+ and CD4+ T cells to the lungs, and were unable to clear the pathogen from their lungs at day 14. At day 14 the MyD88-deficent mice developed a severe, chronic lung inflammation with elevated IL-1beta and IFN-gamma leading to increased mortality, whereas wild-type mice as well as TLR2- or TLR4-deficient mice recovered from acute pneumonia and did not show delayed bacterial clearance. Thus, MyD88 is essential to recognize C. pneumoniae infection and initiate a prompt and effective immune host response against this organism leading to clearance of bacteria from infected lungs.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

Surfactant protein D enhances Pneumocystis infection in immune-suppressed mice

To further determine the role of surfactant protein (SP)-D in the pathogenesis of Pneumocystis pneumonia, a mouse model of transgenic overexpression (OE) of SP-D was studied. These animals produce roughly 30- to 50-fold greater SP-D than their wild-type (WT) counterparts but show no other differences in lung morphology and function. Animals in both the SP-D OE and WT groups were depleted of CD4 lymphocytes with weekly injections of GK1.5 antibody, before Pneumocystis inoculation, and throughout the subsequent infection period. At various time points, mice were killed and analyzed for inflammatory parameters and organism burden. Proinflammatory cytokines in bronchoalveolar lavage fluid were elevated throughout the period of infection, with OE animals exhibiting significantly higher levels of TNF-alpha and macrophage inflammatory protein-2 compared with WT controls. The total number of cells in the lavage fluid was also increased significantly only in the OE group, whereas the cell differential composition demonstrated lymphocyte and eosinophil infiltration in both groups of animals. Significantly, the organism burden was markedly higher in the SP-D OE animals, whereas the WT mice demonstrated little alteration in organism number over the course of infection. These results further indicate that SP-D facilitates the development of Pneumocystis infection and related lung inflammation in an immunosuppressed mouse model.     

TLR4 through IFN-β promotes low molecular mass hyaluronan-induced neutrophil apoptosis

Intratracheal administration of low molecular mass (LMM) hyaluronan (200 kDa) results in greater neutrophil infiltration in the lungs of TLR4(-/-) mice compared with that in wild-type mice. In general, enhanced neutrophil infiltration in tissue is due to cell influx; however, neutrophil apoptosis also plays an important role. We have assessed the effects of TLR4 in the regulation of neutrophil apoptosis in response to administration of LMM hyaluronan. We found that apoptosis of inflammatory neutrophils is impaired in TLR4(-/-) mice, an effect that depends upon the IFN-β-mediated TRAIL/TRAILR system. IFN-β levels were decreased in LMM hyaluronan-treated TLR4-deficient neutrophils. The treatment of inflammatory neutrophils with IFN-β enhanced the levels of TRAIL and TRAILR 2. LMM hyaluronan-induced inflammatory neutrophil apoptosis was substantially prevented by anti-TRAIL neutralizing mAb. We conclude that decreased IFN-β levels decrease the activity of the TRAIL/TRAILR system in TLR4-deficient neutrophils, leading to impaired apoptosis of neutrophils and resulting in abnormal accumulation of neutrophils in the lungs of LMM hyaluronan-treated mice. Thus, TLR4 plays a novel homeostatic role in noninfectious lung inflammation by accelerating the elimination of inflammatory neutrophils.     

Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa

Activation of pulmonary defenses against Pseudomonas aeruginosa requires myeloid differentiation factor 88 (MyD88), an adaptor for Toll-like receptor (TLR) signaling. To determine which TLRs mediate recognition of P. aeruginosa, we measured cytokine responses of bone marrow cells from wild-type mice and mice lacking TLR2 (TLR2(-/-)), TLR4 (TLR4(-/-)), TLR2 and TLR4 (TLR2/4(-/-)), or MyD88 (MyD88(-/-)) to wild-type P. aeruginosa and to fliC P. aeruginosa, which lacks the TLR5 ligand flagellin. Mice also were challenged with aerosolized bacteria to determine cytokine responses, lung inflammation, and bacterial clearance. TNF induction required MyD88 and was absent in TLR2/4(-/-) cells in response to fliC but not wild-type P. aeruginosa, whereas TLR2(-/-) cells exhibited augmented responses. In vivo, TLR4(-/-) mice responded to wild-type P. aeruginosa with reduced cytokine production and inflammation, but intact bacterial clearance, while TLR2(-/-) mice had partially impaired cytokine responses and delayed bacterial killing despite normal inflammation. When challenged with fliC, MyD88(-/-) mice failed to mount early cytokine and inflammatory responses or control bacterial replication, resulting in necrotizing lung injury and lethal disseminated infection. TLR4(-/-) and TLR2/4(-/-) mice responded to fliC infection with severely limited inflammatory and cytokine responses but intact bacterial clearance. TLR2(-/-) mice had partially reduced cytokine responses but augmented inflammation and preserved bacterial killing. These data indicate that TLR4- and flagellin-induced signals mediate most of the acute inflammatory response to Pseudomonas and that TLR2 has a counterregulatory role. However, MyD88-dependent pathways, in addition to those downstream of TLR2, TLR4, and TLR5, are required for pulmonary defense against P. aeruginosa.     

TLR-4 pathway mediates the inflammatory response but not bacterial elimination in E. coli pneumonia

We examined the role of Toll-like receptor (TLR)-4 in modifying the lung inflammatory response and its effects on the bacterial recovery from the lungs following inhaled Escherichia coli in two different strains of TLR-4 mutant mice that are hyporesponsive to LPS. The C57BL/10ScN(tlr4(lps-del)) mice containing a deletion mutation in the TLR-4 gene showed lower proinflammatory cytokine levels, lower lung MPO activity, and less parenchymal and peribronchial inflammation compared with the C57BL/10ScSn mice, a related TLR-4 wild-type substrain. However, the C57BL/10ScN(tlr4(lps-del)) mutant showed lower bacterial recovery in the lungs following inhaled E. coli associated with a rapid but transient increase in air space neutrophil counts at 6 h. In comparison, the C3H/HeJ(tlr4(Lps-d)) mutant mice containing a Pro712His substitution in TLR-4 demonstrated lower proinflammatory cytokine levels, lower lung MPO activity, and lower neutrophil accumulation in the air spaces but showed no differences in the bacterial burden of inhaled E. coli at 6 h, when compared with the TLR-4 wild-type C3H/HeSnJ mice. Thus two different TLR-4 mutants showed attenuated inflammatory responses in the lungs, but the reduced inflammatory responses were not consistently associated with either improved or impaired bacterial elimination from the lungs. Our findings indicate that the inflammatory response to inhaled E. coli is TLR-4 dependent, but bacterial elimination depends on other factors in addition to TLR-4.     

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.     

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.     

Regulation of lung injury and repair by Toll-like receptors and hyaluronan

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.     

Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2

Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3⁺ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2''s role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3⁺ Tregs to the lungs to control inflammation.     

The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice

Toll-like receptors (TLR) induce distinct patterns of host responses through myeloid differentiation factor 88 (MyD88)-dependent and/or -independent pathways, depending on the nature of the pathogen. Pseudomonas aeruginosa is a cause of serious lung infection in immunocompromised individuals and cystic fibrosis patients. The role of the TLR-MyD88 pathway in P. aeruginosa-induced lung infection in vivo was examined in this study. MyD88-/- mice demonstrated an impaired clearance of P. aeruginosa from the lung. Little or no neutrophil recruitment was observed in the airways of MyD88-/- mice following P. aeruginosa lung infection. This observation was associated with a reduced production of inflammatory mediators that affect neutrophil recruitment, including macrophage-inflammatory protein-2, tumor necrosis factor, and interleukin-1beta in the airways of MyD88-/- mice. Similarly, MyD88-/- mice showed inhibited NF-kappaB activation in the lung following P. aeruginosa infection. Interestingly, P. aeruginosa infection induced a 7.5-fold increase of TLR2 mRNA expression in the lungs of MyD88+/+ mice. Furthermore, host responses to P. aeruginosa lung infection in TLR2-/- and TLR4 mutant mice were partially inhibited compared with the responses of respective control mice. Taken together, our results indicate that the MyD88-dependent pathway is essential for the development of early host responses to P. aeruginosa infection, leading to the clearance of this bacterium, and that TLR2 and TLR4 are involved in this process.     

Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo

The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.     

Eosinophils mediate SIgA production triggered by TLR2 and TLR4 to control Ascaris suum infection in mice

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host’s organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles.