Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice

Background

Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.

Methods

BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.

Results

Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.

Conclusion

Smoke induced inflammation does not protect against influenza infection.In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.     

Cigarette smoke exposure aggravates air space enlargement and alveolar cell apoptosis in Smad3 knockout mice

The concept of genetic susceptibility factors predisposing cigarette smokers to develop emphysema stems from the clinical observation that only a fraction of smokers develop clinically significant chronic obstructive pulmonary disease. We investigated whether Smad3 knockout mice, which develop spontaneous air space enlargement after birth because of a defect in transforming growth factor-β (TGF-β) signaling, develop enhanced alveolar cell apoptosis and air space enlargement following cigarette smoke exposure. We investigated Smad3(-/-) and Smad3(+/+) mice at different adult ages and determined air space enlargement, alveolar cell proliferation, and apoptosis. Furthermore, laser-capture microdissection and real-time PCR were used to measure compartment-specific gene expression. We then compared the effects of cigarette smoke exposure on Smad3(-/-) and littermate controls. Smad3 knockout resulted in the development of air space enlargement in the adult mouse and was associated with decreased alveolar VEGF levels and activity and increased alveolar cell apoptosis. Cigarette smoke exposure aggravated air space enlargement and alveolar cell apoptosis. We also found increased Smad2 protein expression and phosphorylation, which was enhanced following cigarette smoke exposure, in Smad3-knockout animals. Double immunofluorescence analysis revealed that endothelial apoptosis started before epithelial apoptosis. Our data indicate that balanced TGF-β signaling is not only important for regulation of extracellular matrix turnover, but also for alveolar cell homeostasis. Impaired signaling via the Smad3 pathway results in alveolar cell apoptosis and alveolar destruction, likely via increased Smad2 and reduced VEGF expression and might represent a predisposition for accelerated development of emphysema due to cigarette smoke exposure.     

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.     

The dendritic cell populations of mouse lymph nodes.

The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4(+)8(-)DEC-205(-), CD4(-)8(-)DEC-205(-), CD4(-)8(+)DEC-205(+)), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.     

Cigarette smoke exposure increases the cell water organization and membrane order of cultured T cells.

In order to determine the effects of cigarette smoke (CS) exposure on the physical properties of cells, NMR water-proton relaxation time (which measures the intracellular water organization) and ESR spin labeling (which measures membrane order) measurements were performed on cultured Jurkat T cells exposed to CS. NMR spin-lattice relaxation time (T1) decreased with CS exposure in a dose-dependent fashion. A significantly depressed T1 value was obtained even when CS was delivered through a filter. Cell viability was not affected in this condition. Superoxide dismutase (SOD) prevented the depression of T1 value. These results suggest that superoxide radicals or subsequently generated species contained in the gas phase of CS increase the intracellular water organization in viable cells. CS exposure also increased the ESR membrane order parameter of nitroxide spin label. These physical characteristic changes may be important in CS-induced cell responses and cytopathology.     

Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin

Osteopontin (OPN) has been reported to enhance the interferon (IFN)-gamma-producing Th1-type T cell response through the induction of interleukin (IL)-12 and the suppression of IL-10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co-inoculation of OPN suppressed the induction of IFN-gamma-producing CD4(+) T cells and T cell proliferative response after the subcutaneous heat-killed Listeria monocytogenes(HKLM) immunization. These results suggest that OPN down-regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow-derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN-treated DC showed a normal antigen-presenting function to an established Listeria-specific Th1-type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM-induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN-mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration.     

Memory T cells are enriched in lymph nodes of selectin-ligand-deficient mice

Fucosyltransferase-IV and -VII double knockout (FtDKO) mice reveal profound impairment in T cell trafficking to lymph nodes (LNs) due to an inability to synthesize selectin ligands. We observed an increase in the proportion of memory/effector (CD44(high)) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44(high)CD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44(high)CD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44(high)CD8 T cells were up to 8-fold higher in LNs of FtDKO mice at day 60. Because wild-type mice treated with anti-CD62L treatment also showed increased frequencies of CD44(high) T cells in LNs, we hypothesized that memory T cells were preferentially retained in, or preferentially migrated to, FtDKO LNs. We analyzed T cell entry and egress in LNs using adoptive transfer of bone fide naive or memory T cells. Memory T cells were not retained longer in LNs compared with naive T cells; however, T cell exit slowed significantly as T cell numbers declined. Memory T cells were profoundly impaired in entering LNs of FtDKO mice; however, memory T cells exhibited greater homeostatic proliferation in FtDKO mice. These results suggest that memory T cells are enriched in LNs with T cell deficits by several mechanisms, including longer T cell retention and increased homeostatic proliferation.     

CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.     

CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells

The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8+ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8+ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Ralpha). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8+ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11chigh dendritic cells. Our results suggest a dual role of CD11chigh dendritic cells as unique APC and cytokine-presenting cells.     

Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study

Background

Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD.

Methods

The extent of dendritic cell infiltration in COPD tissue specimens was determined using immunohistochemical localization of CD83+ cells (marker of matured myeloid dendritic cells), and CD1a+ cells (Langerhans cells). The extent of tissue infiltration with Langerhans cells was also determined by the relative expression of the CD207 gene in COPD versus control tissues. To determine mechanisms by which dendritic cells accumulate in COPD, complimentary studies were conducted using monocyte-derived human dendritic cells exposed to cigarette smoke extract (CSE), and dendritic cells extracted from mice chronically exposed to cigarette smoke.

Results

In human COPD lung tissue, we detected a significant increase in the total number of CD83+ cells, and significantly higher amounts of CD207 mRNA when compared with control tissue. Human monocyte-derived dendritic cells exposed to CSE (0.1-2%) exhibited enhanced survival in vitro when compared with control dendritic cells. Murine dendritic cells extracted from mice exposed to cigarette smoke for 4 weeks, also demonstrated enhanced survival compared to dendritic cells extracted from control mice. Acute exposure of human dendritic cells to CSE induced the cellular pro-survival proteins heme-oxygenase-1 (HO-1), and B cell lymphoma leukemia-x(L) (Bcl-xL), predominantly through oxidative stress. Although activated human dendritic cells conditioned with CSE expressed diminished migratory CCR7 expression, their migration towards the CCR7 ligand CCL21 was not impaired.

Conclusions

These data indicate that COPD is associated with increased numbers of cells bearing markers associated with Langerhans cells and mature dendritic cells, and that cigarette smoke promotes survival signals and augments survival of dendritic cells. Although CSE suppressed dendritic cell CCR7 expression, migration towards a CCR7 ligand was not diminished, suggesting that reduced CCR7-dependent migration is unlikely to be an important mechanism for dendritic cell retention in the lungs of smokers with COPD.     

Regulatory T cells control dendritic cell/NK cell cross-talk in lymph nodes at the steady state by inhibiting CD4+ self-reactive T cells

The CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the control of peripheral tolerance by directly inhibiting conventional T cell proliferative and effector functions. However, the mechanisms by which Treg regulate the homeostasis of lymph nodes remain unclear. In this study, we show in a mouse model that Treg control two major checkpoints dictated by the interaction between self-reactive CD4(+) T cells and resident dendritic cell (DC) in secondary lymphoid organs. First, Treg inhibit the production of CCR5 ligands, limiting the CCR5-dependent recruitment of DC in the lymph nodes. Second, Treg prevent the DC exposure of IL-15Ralpha, markedly interfering in the DC-mediated NK cell proliferation in vivo. Therefore, the DC/T cell autoreactivity leading to NK cell triggering could potentially be controlled by the coinhibition of both IL-15Ralpha and CCR5 in autoimmune disorders in which NK cells play a deleterious role.     

Adrenergic innervation of lymph nodes and the thoracic duct

By means of incubation of slices in 2% solution of glyoxylic acid distribution of adrenergic fibers in the rabbit lymph nodes and in the thoracic lymphatic duct has been studied. Adrenergic fibers get into parenchyma of the lymph nodes via two ways. The first--the perivascular, when the nervous fibers make a plexus and get into the node along the blood vessels, the second--diffuse nervous fibers get together with trabecules in between the lymphoid nodules. The distribution density of the adrenergic fibers is not the same in different groups of the lymph nodes. In the lumbar nodes it is the highest. In the lymph nodes of the cervical part the density of the sympathetic fibers is, as a rule, lower than in the lumbar, but higher than in the axillary nodes. The lowest density of th adrenergic fibers is in the mesenteric, superficial inguinal lymph nodes and in the lymph nodes, situating near the thoracic part of the aorta. In the lymphatic duct wall small amount of adrenergic fibers are revealed, they form a plexus, predominantly in the cranial part.     

Characterizing T cell movement within lymph nodes in the absence of antigen

The recent application of two-photon microscopy to the visualization of T cell movement has presented trajectories of individual T cells within lymphoid organs both in the presence and in the absence of Ag-loaded dendritic cells. Remarkably, even though T cells largely move along conduits of the fibroblastic reticular cell network, they appear to execute random walks in lymphoid organs rather than chemotaxis. In this study, we analyze experimental trajectories of T cells using computer simulations of idealized random walks. Comparisons of simulations with experimental data provide estimates of key parameters that characterize T cell motion in vivo. For example, we find that the distance moved before turning is about twice the distance between intersections in the fibroblastic reticular cell network, suggesting that at an intersection a T cell will turn onto a new fiber approximately 50% of the time. Although the calibrated model appears to offer an accurate representation of T cell movement, it has also uncovered inconsistencies across different experimental data sets.     

Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection

Notch1 is involved in directing cell fate decisions in a variety of developmental scenarios. Extending previous experiments in mice, we generated transgenic rats expressing the intracellular domain of Notch1 in the thymus. Importantly, this leads to sustained expression of the pre-TCR throughout thymocyte development, accompanied by a reduction of alphabetaTCR complexes. In addition, re-expression of RAG-1 and RAG-2 in TCRbeta(+) cells is impaired, and the Valpha repertoire is altered. Consequently, thymocytes in transgenic rats do not undergo positive selection and largely fail to progress to the single positive stage. According to our model, the previously reported effects of Notch1 on the CD4/CD8 cell fate decision may be explained by a differential sensitivity of the two lineages toward altered TCR signaling.     

Mycophenolate mofetil impairs the maturation and function of murine dendritic cells

The immunosuppressive drug, mycophenolate mofetil (MMF), has been successfully introduced in allogeneic transplantation medicine and, more recently, in the treatment of autoimmune skin disorders. MMF inhibits lymphocyte proliferation via a blockade of the enzyme inosine 5'-monophosphate dehydrogenase, an enzyme on which lymphocytes solely depend to generate the purines necessary for DNA/RNA synthesis. To investigate the effects of MMF on cutaneous immune responses, a murine model of contact hypersensitivity (CHS) was used, with oxazolone or trinitrochlorobenzene as a contact allergen. Compared with the respective vehicle, i.p. applied MMF significantly inhibited the elicitation and, surprisingly, the induction of CHS responses. This prompted further studies into the effects of MMF on Ag presentation. Bone marrow-derived dendritic cells (DC) were cultured with GM-CSF and IL-4 in the presence of MMF and were tested for their Ag-presenting capacity. Sensitization and elicitation of CHS and delayed-type hypersensitivity responses by s. c. injected haptenated DC were reduced upon preincubation of DC with MMF. CHS responses were not impaired upon resensitization, indicating that MMF does not induce hapten-specific immunotolerance. In addition, MMF decreased the ability of DC to stimulate allogeneic T cells in MLR assays. Accordingly, flow cytometric analyses revealed a dose-dependent reduction of the expression of CD40, CD80, CD86, I-A, and ICAM-1 on DC with a concurrent reduction of IL-12 production. These data suggest that MMF, in addition to affecting T lymphocytes, directly affects APC, resulting in an impairment of immune responses. They furthermore point to a possible role of inosine 5'-monophosphate dehydrogenase in the maturation of DC.     

Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.     

Limited infiltration of exogenous dendritic cells and naive T cells restricts immune responses in peripheral lymph nodes

Primary CD8 T cell responses in lymph nodes (LN) and protective immunological tumor control are quantitatively limited following immunization with exogenous peptide-pulsed dendritic cells (DC). This arises from two constraints. First, LN are saturated by relatively small quantities of exogenous DC. Second, circulation of new naive T cells into DC-infiltrated LN during the functional lifespan of the DC is negligible. Limits on DC and T cellularity in, and flux through, LN constrain the magnitude of both primary and subsequent recall responses. Enhanced immune responses and tumor control can be achieved using maneuvers to augment LN retention of DC or availability of naive T cells to Ag-presenting DC. These data offer an increased understanding of LN function in general and provide a practical basis for improvements in tumor immunotherapy.     

Cigarette smoke exposure induces ROS-mediated autophagy by regulating sestrin,AMPK, and mTOR level in mice

Many pathological conditions linked to cigarette smoking are caused by the production of reactive oxygen species (ROS). The present study was conducted to analyze the effect of ROS on the lungs of Swiss mice exposed to cigarette smoking, focusing on autophagy-mediated mechanisms, and investigate the involvement of SESN2, AMPK, and mTOR signaling. Mice were exposed to cigarette smoke (CS) for 7, 15, 30, 45, and 60 days; the control group was not exposed to CS. Only mice exposed to CS for 45 days were selected for subsequent N-acetylcysteine (NAC) supplementation and smoke cessation analyses. Exposure to CS increased the production of ROS and induced molecular changes in the autophagy pathway, including an increase in phosphorylated AMPK and ULK1, reduction in phosphorylated mTOR, and increases in SESN2, ATG12, and LC3B levels. NAC supplementation reduced ROS levels and reversed all molecular changes observed upon CS treatment, suggesting the involvement of oxidative stress in inducing autophagy upon CS exposure. When exposure to CS was stopped, there were decreases in the levels of oxidative stress, AMPK and ULK1 phosphorylation, and autophagy-initiating molecules and increase in mTOR phosphorylation. In conclusion, these results suggest the involvement of ROS, SESN2, AMPK, and mTOR in the CS-induced autophagic process in the lung.