Khalil Karimi

Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice.

RATIONALE We have previously demonstrated that oral treatment with live Lactobacillus reuteri can attenuate major characteristics of the asthmatic response in a mouse model of allergic airway inflammation. However, the mechanisms underlying these effects remain to be determined. OBJECTIVES We tested the hypothesis that regulatory T cells play a major role in mediating L. reuteri-induced attenuation of the allergic airway response. METHODS BALB/c mice were treated daily with L. reuteri by gavage. Flourescent-activated cell sorter analysis was used to determine CD4(+)CD25(+)Foxp3(+)T cell populations in spleens following treatment with L. reuteri or vehicle control. Cell proliferation assays were performed on immunomagnetic bead separated CD4(+)CD25(+) and CD4(+)CD25(-) T cells. CD4(+)CD25(+) T cells isolated from, ovalbumin naive, L. reuteri treated mice were transferred into ovalbumin-sensitized mice. Following antigen challenge the airway responsiveness, inflammatory cell influx and cytokine levels in bronchoalveolar lavage fluid of recipient mice were assessed. MEASUREMENTS AND MAIN RESULTS Following 9 days of oral L. reuteri treatment, the percentage and total number of CD4(+)CD25(+)Foxp3(+)T cells in spleens significantly increased. CD4(+)CD25(+) cells isolated from L. reuteri-fed animals also had greater capacity to suppress T-effector cell proliferation. Adoptive transfer of CD4(+)CD25(+) T cells from L. reuteri-treated mice to ovalbumin-sensitized animals attenuated airway hyper-responsiveness and inflammation in response to subsequent antigen challenge. CONCLUSIONS These results strongly support a role for nonantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells in attenuating the allergic airway response following oral treatment with L. reuteri. This potent immuno-regulatory action may have therapeutic potential in controlling the Th2 bias observed in atopic individuals.

Toll-like receptor-4 mediates cigarette smoke-induced cytokine production by human macrophages

BackgroundThe major risk factor for the development of COPD is cigarette smoking. Smoking causes activation of resident cells and the recruitment of inflammatory cells into the lungs, which leads to release of pro-inflammatory cytokines, chemotactic factors, oxygen radicals and proteases. In the present study evidence is found for a new cellular mechanism that refers to a link between smoking and inflammation in lungs.MethodsEmploying human monocyte-derived macrophages, different techniques including FACS analysis, Cytometric Bead Array Assay and ELISA were achieved to evaluate the effects of CS on pro-inflammatory cytokine secretion including IL-8. Then, Toll-like receptor neutralization was performed to study the involvement of Toll-like receptor-4 in IL-8 production. Finally, signaling pathways in macrophages after exposure to CS medium were investigated performing ELISA and Western analysis.ResultsWe demonstrate that especially human monocytes are sensitive to produce IL-8 upon cigarette smoke stimulation compared to lymphocytes or neutrophils. Moreover, monocyte-derived macrophages produce high amounts of the cytokine. The IL-8 production is dependent on Toll-like receptor 4 stimulation and LPS is not involved. Further research resolved the cellular mechanism by which cigarette smoke induces cytokine production in monocyte-derived macrophages. Cigarette smoke causes subsequently a concentration-dependent phosphorylation of IRAK and degradation of TRAF6. Moreover, IκBα was phosphorylated which suggests involvement of NF-κB. In addition, NFκB -inhibitor blocked cigarette smoke-induced IL-8 production.ConclusionThese findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema.

The vagus nerve modulates CD4+ T cell activity

The vagus nerve has a counter-inflammatory role in a number of model systems. While the majority of these anti-inflammatory effects have been ascribed to the activation of nicotinic receptors on macrophages, little is known about the role of the vagus in modulating the activity of other cells involved in inflammatory responses. Here, we demonstrate that following subdiaphragmatic vagotomy of mice CD4(+) T cells from the spleen proliferated at a higher rate and produced more pro-inflammatory cytokines, including TNF and IFN-gamma, upon in vitro stimulation. Cell responses were restored to control levels following the administration of nicotine and the treatment of non-vagotomized animals with a nicotinic receptor antagonist could mimic the effect of vagotomy. Our results suggest that vagal input constitutively down-regulates T cell function through action at nicotinic receptors and the role of the vagus in regulating immune responses is more extensive than previously demonstrated.

Stem cell factor and Interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.

Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages

BackgroundToll-like receptors (TLRs) are present on monocytes and alveolar macrophages that form the first line of defense against inhaled particles. The importance of those cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) has well been documented. Cigarette smoke contains high concentration of oxidants which can stimulate immune cells to produce reactive oxygen species, cytokines and chemokines.MethodsIn this study, we evaluated the effects of cigarette smoke medium (CSM) on TLR4 expression and interleukin (IL)-8 production by human macrophages investigating the involvement of ROS.Results and DiscussionTLR4 surface expression was downregulated on short term exposure (1 h) of CSM. The downregulation could be explained by internalization of the TLR4 and the upregulation by an increase in TLR4 mRNA. IL-8 mRNA and protein were also increased by CSM. CSM stimulation increased intracellular ROS-production and decreased glutathione (GSH) levels. The modulation of TLR4 mRNA and surface receptors expression, IRAK activation, IκB-α degradation, IL-8 mRNA and protein, GSH depletion and ROS production were all prevented by antioxidants such as N-acetyl-L-cysteine (NAC).ConclusionTLR4 may be involved in the pathogenesis of lung emphysema and oxidative stress and seems to be a crucial contributor in lung inflammation.

Enhanced Antitumor Immunity Elicited by Dendritic Cell Vaccines Is a Result of Their Ability to Engage Both CTL and IFNγ-producing NK Cells

Activation of cytotoxic T lymphocytes (CTLs) is a primary goal of many cancer vaccination therapies. We have evaluated two vaccination platforms, dendritic cells (DCs) and recombinant adenoviruses (rAds), for their ability to elicit CTL response and antitumor protection. Although rAd was more potent in CTL priming, DC vaccination provided greater protective and therapeutic antitumor activity. Subsequent analyses ruled out the possibility that the two vaccines elicit qualitatively distinct CTL, and demonstrated instead that DCs could better engage natural killer (NK) cells as an additional effector mechanism. We demonstrated that, although both DCs and rAd can stimulate rapid NK expansion, only DC-activated NK cells are able to produce interferon-γ (IFNγ) and mediate antitumor protection. Moreover, our data showed that exogenously delivered DCs preferentially engaged the Mac-1highCD27high NK subset, thereby suggesting that this NK population plays a predominant role in NK:DC interaction. Interestingly, at least 3 days were required for DC-triggered NK cells to acquire effector functions, indicating that a similar priming process operates between T cells and NK cells. Our results suggest that the nature of the vaccine platform can determine the relative involvement of NK and T cells in antitumor immunity, and that methods to augment NK function should be included in vaccination strategies in order to complement CTL-mediated control of tumor growth.

Stem cell factor and interleukin-4 increase responsiveness of mast cells to Substance P

The response of mast cells (MC) to non-IgE-mediated stimulation is critically dependent on the population of MC examined. The neuropeptide Substance P (SP) has been reported to activate connective tissue-type MC (CTMC), while mucosal MC (MMC) are not activated by SP. We examined the effect of stem cell factor (SCF) plus interleukin-4 (IL-4) on SP-initiated activation of bone marrow-derived MC (BMMC). Mouse MC, derived from a culture of BM cells with IL-3, were subsequently treated with recombinant SCF plus IL-4 for 6 days. Responsiveness to SP was monitored measuring beta-hexosaminidase and lipid mediator release. Histochemical staining, histamine analysis, and granule protease expression were achieved to characterize the cells. In contrast to IL-3 grown cells, SCF/IL-4-exposed cells showed functional responsiveness to release beta-hexosaminidase (42.25% +/- 1.46% at SP concentration of 100 microM) and produce leukotriene C(4) (LTC(4)) (7.4 +/- 1.5 ng/10(6) cells)/prostaglandin D(2) (PGD(2)) (2.0 +/- 0.3 ng/10(6) cells) upon stimulation by SP. The increase in sensitivity of the cells to SP was not due to differentiation into CTMC, as the cells remained heparin negative. Both SCF and IL-4 were needed because SCF or IL-4 alone were insufficient to keep cells viable after 3 to 4 days post coculture. SP-induced secretion from BMMC cultured in medium containing SCF plus IL-4 (25.76% +/- 1.83%) was higher in comparison with cells cultured with SCF plus IL-3 (8.85% +/- 0.68%).These findings indicate that temporal changes in cytokine expression can influence the sensitivity of MC to non-immunologic stimuli. Local cytokine production leading to an increase in MC responsiveness to SP and inducing secretion of granule content and lipid generation may, therefore, propagate and worsen inflammatory conditions.

Evidence for neuronal expression of functional Fc (ε and γ) receptors

To the Editor: The Fc receptor family plays a key role in adaptive immunity through the binding of immunoglobulin antibodies that recognize an immune insult and elicit an inflammatory response leading to its clearance. Dysregulation of this receptor family may have untoward consequences that result in autoimmune and allergic diseases. Many of these diseases seem to involve the nervous system and are exacerbated by stress or other neurological challenges. Recently, the presence of Fc receptors was uncovered on dorsal root ganglion neurons and suggested an IgG and possibly IgE-mediated activation of neurons 1–3. We set out to more extensively explore which Fc receptors might be expressed in neurons, and whether they were functional and able to transmit signals to interconnected neurites in vitro and in vivo Messenger RNA was isolated from a highly pure culture of mouse superior cervical ganglion (SCG) neurons 4 and expression of Fc receptor transcripts assessed by reverse transcriptase-polymerase chain reactions (RT-PCR) using specific primers for Fcγ and Fce family members. Fig. 1,a demonstrates the presence of transcripts for the immunoglobulin-binding α chain of FcγRI, II, III, and IV in three individual SCG neuron mRNA preparations. A small amount of the transcript for the low affinity IgE receptor (FceRII or CD23) was also detected relative to that seen in B cells, known to express this receptor. FcγRI transcripts were detected in bone marrow derived mouse mast cells but these levels were less than seen in the neurons. This observation and the inability to detect mMCP-6mRNA in either Balb/c or Bl6 mice, together with the absence of CD23 transcripts in both neurons and mast cells provided confidence that the observed Fc receptor transcripts in neurons was not a result of mast cell contamination of cultures. Figure 1 mRNA expression of the subunits of FceRI, FcγRI-IV, and CD23 in SCG neurons. mRNA was collected from three (1–3) SCG neuronal cultures and expression of the indicated mRNA was measured by RT-PCR. (a) Expression of the α ... We also unexpectedly observed the presence of transcripts for the α, β, and γ chains of the high affinity IgE receptor (FceRI) (Fig. 1,b). While the trimeric form (αγ2) of this receptor has been described in cells other than mast cells or basophils (such as in human Langerhans cells 5), the expression of the tetrameric form (αβγ2) was previously thought to be limited to these pro-inflammatory cells. The trimeric FceRI shows weak calcium signals when compared to the tetrameric form due to the absence of the FceRIβ in the former 6. To determine if the FceRI was expressed on the cell surface of SCG neurons, cells were incubated with IgE and with an antibody to the neuronal specific protein gene product (PGP) 9.5 (which encodes a neuronal ubiquitin C-terminal hydrolase not found on glia) and binding visualized with a fluorescent secondary antibody. IgE was detected on the neuronal cell surface (Fig. 2,a). While the presence of PGP9.5 was most evident in the cell body, IgE binding was detected in plasma membrane of both the cell body and neurite extensions. As shown in Figs. 2,b–d, the expression of FceRI was further confirmed by detection of the α, β, and γ chains of this receptor. Figure 2 Neurons were sensitized with IgE and incubated overnight with antibody against IgE (a, left panel) or with an antibody of unknown specificity (a, right panel). Staining with the neuron specific marker PGP9.5 (b–c, left panel in red) and FceRIα ... Scorpion venom is a known potent selective activator of neurons and elicits a rapid rise in intracellular Ca2+ (Fig. 3,a). To test the functionality of FceRI expressed on neurons, SCG neurons sensitized with DNP-specific IgE were challenged with DNP-HSA (Ag) and a rapid rise in intracellular Ca2+ was observed (Fig. 3,a). No changes in intracellular Ca2+ were observed when serum albumin alone was used as Ag or when cells were not sensitized with IgE (data not shown). FcγRIV was recently described to bind the IgEb allotype but does not recognize the IgEa allotype 7, whereas FceRI binds both allotypes. We excluded that IgE/Ag-mediated calcium responses might occur through FcγRIV, by use of both a IgEa and IgEb allotypes. Both IgE allotypes similarly elicited calcium responses (Fig. 3,a). Addition of Ag elicited a relatively uniform Ca2+ responses in the stimulated cell population (Fig. 3,b). . Based on the high affinity binding of monomeric IgE (was not removed by washing the cells) and the dose response to Ag (Fig. 3, D), we could also exclude the involvement of CD23. Figure 3 (a) Kinetics of [Ca2+]i increase upon stimulation with scorpion venom (SV, Control) or with anti-DNP IgEa,/b sensitized neurons following Ag stimulation (10 ng/ml). The arrows indicate the time of addition of the stimulus. (b) Confocal image of calcium ... Thus, the findings demonstrate the presence of functional high affinity Fce receptors on SCG neurons. Since FcγRIII is known to activate mast cells, its functionality was also tested. SCG neurons sensitized with DNP-specific IgG1, which preferentially binds FcγRII and III but weakly to FcγRI and not to FcγRIV 8, showed modest increases in intracellular Ca2+ that increased with a large dose of Ag (Fig. 3,c). Both IgE and IgG-mediated responses were concentration dependent and a Ca2+ response (Fig. 3,d) was not elicited in all challenged neurons. Moreover, as expected (given the weak binding of monomeric IgG1 to Fcγ receptors), increased responsiveness via IgG required much higher concentrations of Ag than for IgE. To test if the Ca2+ signals elicited by FceRI stimulation could be transferred to interconnected neurites, we explored whether Ca2+ rises might be elicited in neighboring neurites after Ag challenge of an IgE sensitized cell body or neurite. Using a spritzer micro-pipette, antigen was puffed directly onto a neuronal cell body, causing an instantaneous (<5 sec) increased fluorescence in that cell body which moved from there to the connected neurites and propagated to the neighboring cell bodies and neurites (Fig. 4). These, findings showed that FceRI stimulation causes communication among interconnected neurites. To extend these findings to a more physiological setting, we explored whether neurons from the highly innervated intact jejunum 9 would respond to an FceRI stimulus. After placement of a micropipette spritzer (dotted lines shown in Fig. 5,a and g) on a large myenteric plexus ganglion neuron (plain lines shown in Fig. 5,a and g), the anti-DNP IgE sensitized plexus was challenged with Ag. Challenge with a spritz of 1 μg of Ag gave robust calcium responses in adjacent neurons along the nerve fiber in sensitized (2/2) but not in non-sensitized mice (0/3). Repeated spritzes of non-conjugated HSA at this concentration elicited no responses (0/2) (data not shown). To exclude possible mast cell involvement in the transmission of these robust signals, we conducted similar experiments in mast cell deficient W/Wv mice and in their wild type control WBB6F1. In W/Wv mice (3/3) detectable intracellular calcium increases were observed upon Ag challenge (Fig. 5,a–c). No calcium signal was seen when the same ganglion was first challenged with HSA alone (Fig. 5,d–f). The wild type littermates (WBB6F1) responded positively (3/3), upon challenge with specific Ag (Fig. 5,g–i) but gave no response to HSA alone. These findings confirm that the observed signal transmission by FceRI was not likely caused by mast cells and demonstrate the in vivo presence of functional FceRI on jejunal neurons, since sham sensitization in vivo prior to an ex vivo challenge yielded no response to Ag challenge. Figure 4 Activation of interconnected neurites by FceRI stimulation of a single neuronal cell body. (a) Bright field image of neuron network showing relation of Ag-containing spritzer to neuronal cell body sensitized with IgE anti-DNP. (b) At time zero, ... Figure 5 (A–I) Myenteric ganglion calcium imaging. Spritzer (internal bore, 40 μm) is indicated by dotted lines and myenteric plexus by solid lines. (a–c) Anti-DNP IgE sensitized myenteric neurons were imaged in mast cell-deficient W/W ... It is well known that sensory nerves may participate in hypersensitivity reactions, a process known as neurogenic inflammation and several lines of evidence support the notion that sensory nerves may play an important role in cutaneous, lung, GI and joint inflammatory diseases. Here we now demonstrate that functionally active FceRI is expressed on SCG and myenteric plexus neurons. The discovery of functional Fce and Fcγ receptors on nerves clearly shows that this biological compartment is able to respond to the direct stimulus of antibody-antigen interactions. Our findings define an independent neuronal (non-mast cell/non-basophil) compartment with probable involvement in allergic and possibly other diseases.

Cigarette smoke stimulates the production of chemokines in mast cells.

Chronic obstructive pulmonary disease is a major health problem and will become the third largest cause of death in the world by 2020. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular, cigarette smoke (CS), causes the progressive airflow limitation, in which macrophages and neutrophils are attracted by chemokines, leading to oxidative stress, emphysema, small airways fibrosis, and mucus hypersecretion. Smoking is also associated with an increase in mast cell numbers in bronchial mucosa. This study was conducted to determine the direct effects of CS on mast cell function, using murine bone marrow‐derived mast cells (BMMC) as an in vitro model. BMMC were cultured from BALB/cBy mice for 3 weeks. Cells were treated with CS medium (CSM) for 30 min or 16 h. The effects of CSM on mast cell degranulation and chemokine production were measured. Moreover, we investigated the effect of CSM on IκB‐α degradation and p38, Erk1/2, p65, and CREB expression by Western blotting. We found that CSM stimulated the release of chemokines in a noncytotoxic manner but did not induce mast cell degranulation. CSM induced phosphorylation of Erk1/2, p38, and CREB and increased translocation of p65 without degradation of IκB‐α NF‐κB in mast cells. The induction of chemokine production by CSM in mast cells could promote and prolong the inflammatory process. Our observations suggest that mast cells may contribute to the pathogenesis of emphysema through a direct effect of CS on the production of proinflammatory chemokines.

Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB‐1, has potent immunoregulatory effects, and changes nerve‐dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB‐1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat‐shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyers patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4+25+Foxp3+ T cells. JB‐1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C‐type lectin receptors specific intercellular adhesion molecule‐3 grabbing non‐integrin‐related 1 and Dectin‐1, as well as TLR‐2 and ‐9. JB‐1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB‐1 on in situ patch‐clamped enteric neurons. GroEL and anti‐TLR‐2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria‐neuron and ‐immune communication. Together these results offer a mechanistic explanation of how Gram‐positive commensals and probiotics may influence the hosts immune and nervous systems.—Al‐Nedawi, K., Mian, M. F., Hossain, N., Karimi, K., Mao, Y.‐K., Forsythe, P., Min, K. K., Stanisz, A. M., Kunze, W. A., Bienenstock, J. Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems. FASEB J. 29, 684‐695 (2015). www.fasebj.org