Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.     

Neonatal B cells suppress innate toll-like receptor immune responses and modulate alloimmunity

It has been known for decades that neonates are susceptible to transplant tolerance, but the immunological mechanisms involved remain to be fully elucidated. Recent evidence indicates that the maturation state of DCs responding to an allograft may have a profound impact on whether immunity or tolerance ensues. Given that TLR activation is a key process leading to DC maturation, we hypothesized that DCs from neonates have defective TLR immune responses. Contrary to our hypothesis, we found that murine neonatal DCs demonstrated enhanced TLR responses in comparison to adult counterparts in vitro. However, we found that neonatal B cells possess unique immunoregulatory functions as they impaired DC responses to TLR activation in an IL-10-dependent fashion. Functionally, we demonstrated that TLR-activated neonatal, but not adult, B cells impaired Th1, but not Th2, T cell alloimmune responses in vitro and in vivo, in models of alloimmune priming and allotransplantation. We conclude that neonatal B cells possess unique immunoregulatory properties that inhibit DC function and modulate alloimmunity in our murine experimental systems.     

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.     

Serotonin regulates innate immune responses of colon epithelial cells through Nox2-derived reactive oxygen species

Changes in serotonin (5-hydroxytryptamine, 5-HT) content in the gut of patients with inflammatory bowel disease (IBD) and animal models of colitis suggest an important role of 5-HT in the pathogenesis of IBD. In this study, we examined the role and mechanism of action of 5-HT in the inflammatory response of colon epithelial cells in vitro and in vivo. In colon epithelial cells (CCD 841, HT-29, Caco-2), direct application of 5-HT induced production of reactive oxygen species (ROS) and monocyte–epithelial adhesion, an initial event of inflammation, which were blocked not only by 5-HT receptor antagonists (tropisetron, RS39604, and SB269970), antioxidants (ascorbic acid, apocynin), and various inhibitors of NADPH oxidase (DPI), CREB (KG-501), and NF-κB (PDTC), but also by transfection with Nox2 siRNA. Nox2-derived production of ROS corresponded with the rapid and brief activation of Rac. In addition, 5-HT induced Nox2, p67^phox, and Duox2 without altering the level of Nox1 or Duox1 in colon epithelial cells, and silencing of Nox2 suppressed 5-HT-induced Duox2 increase. 5-HT also induced an increase in the expression of MCP-1, IL-8, and ICAM-1 and a decrease in E-cadherin expression. Exogenous application of 5-HT to rat colon through the rectum caused a minimal level of inflammation, which was demonstrated by histological examination, MPO activity, and inflammatory cytokine induction. However, 5-HT combined with a low dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS), the level of which caused a minimal level of colitis, exaggerated colon inflammation accompanied by much more enhanced induction of inflammatory cytokines, IL-6, IL-8, and MCP-1, indicating that colon epithelial cells directly exposed to 5-HT are primed toward inflammation. In the colon at the lesion site, treatment with 5-HT resulted in an increase in the level of epithelial Nox2 but not of constitutively expressed Nox1, which is the opposite result of TNBS treatment. Furthermore, 5-HT treatment of Nox2-knockout mice did not induce colon inflammation, in contrast to 5-HT-treated wild-type mice. The results demonstrate that colon epithelial cells directly exposed to 5-HT are primed for inflammatory reactions, which is an important innate immune response, and the underlying mechanism for the priming is associated with Nox2-activated signaling pathways, including ERK/p38 MAPK, NF-κB, and CREB.     

Colostrum and bioactive,colostral peptides differentially modulate the innate immune response of intestinal epithelial cells

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mIC_c12) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C₁₈ RP‐HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mIC_c12 cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll‐like receptors (TLR). Whereas intact colostrum generally reduced TLR‐mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.     

The WAP protein Trappin-2/Elafin: a handyman in the regulation of inflammatory and immune responses

Trappin-2/Elafin is a potent serine protease inhibitor which prevents excessive damage under inflammatory status. This "alarm-antiprotease" is locally expressed by epithelial cells and immune cells such as macrophages and γδ T cells. It has also been proven to modulate a wide range of parameters that are critical for the inflammation process like modulating the NFκB pathway, cytokine secretion and cell recruitment. In addition, Trappin-2/Elafin was shown to possess anti-microbial properties against different classes of pathogens including viruses, fungi and bacteria. Studies also linked Trappin-2/Elafin to either susceptibility or protection against inflammatory disease and infections, even though the mechanisms remains poorly understood. This review will discuss some of the pleiotropic effects displayed by Trappin-2/Elafin, and the properties that could be used to prevent infection or to protect against inflammation.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24h elicited a marked increase in mRNA expression for IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway, although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components.     

肠粘膜上皮细胞在天然免疫中的作用

粘膜免疫是机体防御系统的主要成分。致病性细菌侵入机体后,首先遭遇到天然免疫的抵抗,随后产生获得性免疫,两共同执行机体的防御功能,消灭入侵细菌。最近的研究表明上皮细胞对细菌感染有重要的免疫调节作用,在天然免疫与获得性免疫防御机制中起重要作用。本重点介绍肠上皮细胞在天然免疫中的作用。     

Dendritic cells and C-type lectin receptors: coupling innate to adaptive immune responses

Dendritic cells (DCs) have an important function in the initiation and differentiation of immune responses, linking innate information to tailored adaptive responses. Depending on the pathogen invading the body, specific immune responses are built up that are crucial for eliminating the pathogen from the host. Host recognition of invading microorganisms relies on evolutionarily ancient, germline-encoded pattern recognition receptors (PRRs) that are highly expressed on the cell surface of DCs, of which the Toll-like receptors (TLRs) are well characterized and recognize bacterial or viral components. Moreover, they bind a variety of self-proteins released from damaged tissues including several heat-shock proteins. The membrane-associated C-type lectin receptors (CLRs) recognize glycan structures expressed by host cells of the immune system or on specific tissues, which upon recognition allow cellular interactions between DCs and other immune or tissue cells. In addition, CLRs can function as PRRs. In contrast to TLRs, CLRs recognize carbohydrate structures present on the pathogens. Modification of glycan structures on pathogens to mimic host glycans can thereby alter CLR interactions that subsequently modifies DC-induced polarization. In this review, we will discuss in detail how specific glycosylation of antigens can dictate both the innate and adaptive interactions that are mediated by CLRs on DCs and how this balances immune activation and inhibition of DC function.     

Escherichia coli ghosts promote innate immune responses in human keratinocytes

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.     

The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.     

Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.     

Oxidized human beta2-glycoprotein I: its impact on innate immune cells

Beta2-glycoprotein I (β2-GPI), an abundant 50 kDa plasma glycoprotein, is the most common target for antiphospholipid antibodies (aPLs). These autoantibodies are associated with thrombotic events in patients with anti-phospholipid antibody syndrome (APS) and systemic lupus erythematosus (SLE) and are proatherogenic. β2-GPI can also stimulate a vigorous adaptive cellular immune response in these patients. Although much is known about β2-GPI as a cofactor in autoimmune diseases, crucial information is still lacking to clarify why this abundant self plasma protein is the target of autoimmune responses. Throughout the years, a remarkable number of theories have been proposed to explain how the immune system recognises self. On the basis of a large variety of epidemiological, clinical and experimental evidence, it has been suggested that an unfortunate interplay of genetic susceptibility and environmental factors may play an important role in generating an abnormal immune response. Among the environmental factors, oxidative stress is one of the major events causing protein structural modifications, thus inducing the appearance of neo/cryptic epitopes of β2-GPI able to activate the immune system. In particular, oxidized β2-GPI is able to induce phenotypic and functional maturation of dendritic cells which represent the link between innate and adaptive immunity. Chronic activation of autoimmune reactions against this self protein modified by oxidative events may contribute to local and systemic inflammation, thus sustaining endothelial dysfunction in patients with APS, SLE and cardiovascular diseases. The role of oxidative stress in β2-GPI-mediated immune response is described in the light of our research experience and of relevant literature emerging in the field.     

Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system

Yeast derivatives including yeast cell wall components are promising alternatives to antibiotics with respect to the promotion of health and performance in livestock, based on their capacity to bind enteropathogenic bacteria and to beneficially modulate the immune system. However, these mode(s) of action both in vitro and in vivo are still not well understood. Furthermore, standardization and reproducibility of in vitro techniques (microbiology, cell culture assays) are critical features for the application of yeast derivatives as well as for the proof of effectiveness. Yeast cell wall products are suggested as anti-adhesive agents and are thus proposed to prevent attachment of certain intestinal bacteria by providing alternative adhesion sites to enterobacteria, which contain mannose-specific type I fimbriae such as Escherichia coli or Salmonella spp. and which is well documented. Various in vitro assay techniques have become of paramount importance for biotechnological research since they allow for determination and quantification of potential mode(s) of action. However, in vitro assays may be criticized by product end users as not accurately reflecting in vivo responses. Pro and cons of different assays and their bias will be discussed specifically regarding yeast cell wall components and adhesion of enteropathogenic bacteria. Immunomodulation is a therapeutic approach intervening in auto-regulating processes of the defense system. Yeast derivatives such as beta-glucans are proposed to interact with cells of the innate immune system by receptor recognition. Controversial data in literature and mode(s) of action are reviewed and discussed here.     

Leukotrienes: underappreciated mediators of innate immune responses

Leukotrienes are bronchoconstrictor and vasoactive lipid mediators that are targets in the treatment of asthma. Although they are increasingly recognized to exert broad proinflammatory effects, their role in innate immune responses is less well appreciated. These molecules are indeed synthesized by resident and recruited leukocytes during infection. Acting via cell surface G protein-coupled receptors and subsequent intracellular signaling events, they enhance leukocyte accumulation, phagocyte capacity for microbial ingestion and killing, and generation of other proinflammatory mediators. Interestingly, a variety of acquired states of immunodeficiency, such as HIV infection and malnutrition, are characterized by a relative deficiency of leukotriene synthesis. The data reviewed herein point to leukotrienes as underappreciated yet highly relevant mediators of innate immunity.