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TGF-β prevents eosinophilic lung disease but impairs pathogen clearance
Microbes and Infection 7 (2005) 365–374 www.elsevier.com/locate/micinf
Original article
TGF-b prevents eosinophilic lung disease but impairs pathogen clearance Andrew Evan Williams a,*,1, Ian Robert Humphreys a,*,1, Megan Cornere a, Lorna Edwards a, Aaron Rae b, Tracy Hussell b a
Kennedy Institute of Rheumatology, Charing Cross Campus, Imperial College London, London W6 8LM, UK b Centre for Molecular Microbiology and Infection, Department of Biological Sciences, South Kensington, Imperial College London, London SW7 2AZ, UK Received 5 August 2004; accepted 10 November 2004 Available online 19 March 2005
Abstract Respiratory infections are the third leading cause of death worldwide. Complications arise directly as a consequence of pathogen replication or indirectly due to aberrant or excessive immune responses. In the following report, we evaluate the efficacy, in a murine model, of nasally delivered DNA encoding TGF-b1 to suppress immunopathology in response to a variety of infectious agents. A single nasal administration suppressed lymphocyte responses to Cryptococcus neoformans, influenza virus and respiratory syncytial virus. The suppression did not depend on the phenotype of the responding T cell, since both Th1 and Th2 responses were affected. During Th2-inducing infection, pulmonary eosinophilic responses were significantly suppressed. In all cases, however, suppressed immunity correlated with increased susceptibility to infection. We conclude that nasal TGF-b treatment could be used to prevent pulmonary, pathogen-driven eosinophilic disease, although anti-pathogen strategies will need to be administered concordantly. © 2005 Elsevier SAS. All rights reserved. Keywords: Viral; Cytokines; Inflammation; Lung; Mouse
1. Introduction TGF-b plays a central role in the regulation of inflammatory responses including cell proliferation, extracellular matrix deposition, cell migration and differentiation [1]. It is regarded as an immunosuppressive factor and can down-regulate both Th1 and Th2 type responses in addition to its profibrotic action [2–4]. Three isoforms exist in mammals (TGF-beta 1, -beta 2 and -beta 3), each isoform being encoded on different chromosomes [4]. TGF-b is produced by most cell types and is secreted in a latent form, bound to latency-associated peptide (LAP), requiring dissolution for biological activity [5,6]. The activation of latent TGF-b is a crucial regulatory step in con-
Abbreviations: APC, antigen-presenting cell; BAL, broncho-alveolar lavage; IFN-c, interferon-c; IL-4, interleukin-4; RSV, respiratory syncytial virus; TGF-b, transforming growth factor-b; Th, T helper; TNF, tumour necrosis factor. * Corresponding authors. Tel.: +44 20 8383 4769; fax: +44 20 8383 4499. E-mail address: [email protected] (A.E. Williams). 1 These authors contributed equally to this work. 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.11.012
trolling inflammation. LAP may re-associate with TGF-b, rendering it inactive [5]. The 45-kDa protein, decorin, is also expressed in pulmonary tissues and may play a role in counteracting the profibrotic actions of TGF-b [7]. The role of TGF-b is clearly identified in knockout mice, which generate uncontrolled inflammatory responses in several organs. Production of TGF-b is thought to mediate oral tolerance [8,9] and administration of recombinant TGF-b1 suppresses encephalomyelitis, inflammatory bowel disease and uveitis [8,10–12]. However, such therapy requires repeated administration of substantial quantities of protein [10]. TGF-b can suppress both Th1 and Th2 cells via cell cycle inhibition, and it acts to directly antagonize interleukin-4 (IL-4) and IL-12 signaling pathways [13,14]. Several studies report a deviation of T cells towards a Th2 phenotype under the influence of TGF-b. Immune privilege in the eye is mediated by TGF-b and leads to Th2 cytokine predominance [15]. TGF-b plays a critical role in mucosal immune homeostasis; preventing detrimental inflammatory responses, while promoting non-inflammatory dimeric IgA. However, this balance is affected by the presence of an infection that requires
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the induction of an immune response for eradication. Resistance of certain mouse strains to visceral leishmaniasis [16,17] and infection with Plasmodium species [17] depends on low production of TGF-b and high levels of interferon-c (IFN-c). Recombinant TGF-b enhances the survival of Salmonella typhimurium in the mouse gut [18], presumably by downregulating inflammation. However, the role of TGF-b at other mucosal sites is not well understood. Its primary role in the immunopathogenesis of the advanced forms of pulmonary tuberculosis is clear [19]. Higher levels of TGF-b are observed in pulmonary macrophages [20], tuberculosis pleurisy [21] and during in vivo infection [19] possibly via engagement of Toll-like receptor 2 [22]. However, its importance for other pulmonary infections is not well understood. In the following report, we determine that overexpression of TGF-b, through the administration of plasmid DNA expressing the gene for TGF-b1, in a variety of lung infection models reduces inflammatory diseases. However, the ability to clear the pathogen was reduced, and hence, illness was enhanced. Three different infection models were tested so as to include both viral and non-viral disease and Th1-versus Th2-driven immunopathology. Influenza virus is a causative agent of both epidemic and pandemic respiratory viral illness, with over 26,000 deaths attributed to the last outbreak in Britain alone [23]. Though pivotal for viral clearance, overexuberant, Th1-driven T cell responses result in airway occlusion [24,25] and contribute significantly to pathology [26]. Infection of BALB/c mice with influenza reflects many of the features of the human disease, with cachexia, weight loss, fever and pulmonary occlusion. Respiratory syncytial virus (RSV) is another significant respiratory pathogen that causes bronchiolitis in infants [27]. Children hospitalized with bronchiolitis often suffer recurrent wheezing and are frequently diagnosed as asthmatic [28]. There is strong evidence that viral bronchiolitis is a T cell-mediated immunopathological condition [29,30]. In the BALB/c mouse, primary intranasal infection with RSV causes mild self-limiting illness characterized by Th1-driven T cell infiltration. The fungus Cryptococcus neoformans is a significant respiratory pathogen causing pulmonary eosinophilia in immunocompetent hosts [31] and summer-type hypersensitivity pneumonitis in Japan [32]. In immune-deficient patients, dissemination of the pathogen occurs due to the inability of the host to limit the infection [33,34]. The C57BL/6 model of C. neoformans infection mimics the situation in humans and is characterized by Th2driven pulmonary eosinophilia. Lung administration of a DNA plasmid provided a useful method of expressing an active form of TGF-b1. TGF-b1 has multiple effects on a variety of inflammatory cells. The results demonstrate that TGF-b moderates both Th2-driven eosinophilic and Th1 cell-mediated pathology but that this antiinflammatory treatment allows unchecked pathogen replication. We conclude that TGF-b treatment is a viable strategy for preventing pulmonary inflammation but anti-pathogen therapy will need to be administered concurrently.
2. Materials and methods 2.1. Mice and pathogen stocks Eight- to 10-week-old female BALB/c and C57BL/6 mice (Harlan Olac Ltd., Bicester, UK) were kept in specifiedpathogen-free conditions, according to institutional and Home Office (UK) guidelines. Influenza A strain X31 (haemagglutinin [HA] titre 1024) was a kind gift from Dr. Alan Douglas (National Institute for Medical Research, London, UK). RSV was grown in HEp-2 cells and assayed for infectivity as previously described [35]. All stocks were free of mycoplasma, assayed by DNA hybridization (Gen-Probe Inc., San Diego, CA). C. neoformans 52 was obtained from the American Type Culture Collection (no. 24067, Rockville, MD, USA). For infection, yeast was grown to stationary phase (48–72 h) at room temperature on a shaker in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, USA). The cultures were washed in saline, counted on a haemocytometer and diluted in sterile non-pyrogenic saline to the required infective dose. The yeast were plated on Sabouraud agar plates (Difco), and colonies were counted after 48 h culture at room temperature to assess viability. 2.2. Plasmid preparation Plasmid DNA, either pCI-neo (control plasmid with no insert) or pCI-neo containing the gene for murine TGF-b1 was a kind gift from Barry Rouse (Tennessee, USA). DH5a competent cells (Invitrogen, UK) were transformed with plasmid DNA under ampicillin selection. Experimental quantities of DNA were attained using endotoxin-free giga preparation (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The integrity of the plasmid was analyzed on a 0.6% agarose gel in the presence of ethidium bromide. Expression levels of TGF-b1 in murine lung tissue, following a single administration, have been tested previously [36]. Expression of TGF-b from plasmid DNA was assayed by transfection of COS-7 cells, followed by specific immunohistochemistry and TGF-b specific ELISA (Becton Dickinson). 2.3. Experimental procedure Mice were administered either 50 µl PBS or 100 µg of either control or TGF-b-expressing plasmid DNA in 50 µl PBS intranasally (i.n.), 1 week prior to i.n. infection with 103 cfu C. neoformans, 106 pfu RSV or 50 HA units of influenza virus. C. neoformans infections, and RSV and influenza infections were performed in C57BL/6 and BALB/c mice, respectively. Mice were monitored daily, and weight loss was measured throughout infection. Mice were killed at various time points by the injection of 3 mg pentobarbitone and exsanguination of the femoral vessels. 2.4. Cell recovery Broncho-alveolar lavage (BAL) fluid, lung tissue and serum were obtained from each mouse as described previ-
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ously [37]. In brief, lungs were inflated six times with 1.5 ml of Eagle’s minimum essential medium (Sigma) containing 10 mM EDTA and kept on ice. One-hundred milliliters of this BAL fluid was cytocentrifuged onto glass slides, fixed in methanol and stained with haematoxylin and eosin (H and E; Sigma, Dorset UK). The remainder was centrifuged, the supernatant decanted, and the cell pellet re-suspended to 1 ×106 cells per ml in RPMI (Sigma) containing 10% fetal calf serum (FCS), 2 mM/ml L -glutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin (R10F). Lung tissue was homogenized to obtain single cell suspensions, the red blood cells were lysed and the cell pellet re-suspended at 1 × 106 cells per ml in R10F. 2.5. Enumeration of eosinophils Granulocytes were enumerated by flow cytometry based on their size (forward scatter) and granularity (side scatter). From cytopsin preparations, the proportion of eosinophils was determined by their distinctive nuclear morphology and the presence of acidophilic red granules. Neutrophils were identified by the presence of multi-lobed nuclei and the absence of acidophilic granules. 2.6. Flow cytometry Cells (1 × 106) obtained from the airways or the lungs were stained with the following antibody combinations. (1) AntiCD45RB FITC, anti-CD4 allophycocyanin (APC) and antiCD8 PerCP (all BD Pharmingen, Heidelberg, Germany). (2) To detect intracellular cytokines, 1 × 106 cells were incubated with 50 ng/ml PMA (Sigma-Aldrich), 500 ng ionomycin (Calbiochem, Nottingham, UK) and 10 µg/ml brefeldin A (Sigma) for 4 h at 37 °C. Cells were stained with antiCD4 APC and anti-CD8 PerCP on ice for 30 min, washed and then fixed in 2% formaldehyde for 20 min at room temperature. Cells were permeabilized with 0.5% saponin in PBS containing 1% BSA and 0.1% azide for 10 min. A combination of anti-IFNc FITC and anti-IL-4 PE antibodies (BD Pharmingen), diluted in saponin buffer, was then added to the cells. After 30 min, cells were washed once in saponin buffer and twice in PBS containing 0.1% azide and 1% BSA. Samples were analyzed on a FACSCaliber (BD Biosciences, Belgium), collecting data on at least 30,000 lymphocytes. 2.7. Influenza plaque assays Clearance of influenza was assessed from lung homogenates following virus challenge. Homogenized cells were freeze-thawed three times, centrifuged at 4000 × g, and supernatants titrated in doubling dilutions on Madin–Darby canine kidney cell monolayers in flat-bottomed 96-well plates. After incubation at room temperature for 3 h, samples were overlayed with methycellulose and incubated for 72 h at 37 °C. Cell monolayers were washed and incubated with antiinfluenza antibody (Serotec UK, Oxford, UK), followed by
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anti-mouse-HRP (Dako Ltd., Cambridge, UK), and infected cells were detected using 3-amino-9-ethylcarbazole (AEC, Sigma). Infectious units were enumerated by light microscopy, and total plaque-forming units per lung were quantified (number of plaques _ dilution factor _ total volume of lung homogenate). 2.8. RSV plaque assay Infectivity was determined in lung homogenates by infection of HEp-2 cell monolayers for 2 h at 37 °C in serum-free RPMI, prior to overlaying with 150 µl R10F. After 48 h, the monolayer was washed in PBS with 1% BSA and fixed with 100 µl per well of methanol and 0.6% H2O2 for 20 min. Cells were then stained for anti-RSV-HRP (Biogenesis, Poole, Dorset, UK) for 1 h, washed twice with BSA/PBS, and plaques visualized by addition of AEC substrate for 30 min. Plaques were enumerated by light microscopy. 2.9. Enumeration of C. neoformans burden Lungs were homogenized by passage through 100-µm cell strainers. Homogenate (100 µl) was diluted in sterile PBS and incubated at room temperature for 48 h on Sabouraud dextrose agar plates (Sigma). The total colony-forming units per lung was then determined (number of colonies × dilution factor × original cell suspension volume). 2.10. C. neoformans specific IgE Heat-killed C. neoformans (2 × 105 cfu/ml) in PBS was used to coat 96-well micro-titre plates overnight at room temperature on a shaker. After blocking with 3% BSA/PBS for 2 h at room temperature, BAL fluid diluted 1:2 in BSA/PBS was added for a further hour at room temperature. Bound IgE antibody was detected with anti-IgE-HRP (Serotec UK) in BSA/PBS. Plates were washed again, and bound antibody detected with o-phenylenediamine dihydrochloride (OPD, Sigma) substrate. The reaction was stopped with 2 M sulfuric acid. Known IgE standard concentration was unavailable, therefore, differences between treatment groups were measured by absorbance at 490 nm, and background measured as a value of 0.2 using uninfected mouse BAL fluid. 2.11. Analysis of FITC-dextran uptake BALB/c mice were i.n. administered 100 µg of plasmid DNA (TGF-b or control) or PBS in 50-µl volume, twice at weekly intervals. Seven days later, mice were i.n. infected with 50 HA units of influenza virus. One day later, 50 µg of FITC-dextran in 50 µl was administered i.n. At various time points, lung, BAL and mediatinal lymph nodes were sampled for flow cytometry. Cells expressing FITC and anti-CD11b PE (BD Pharmingen) were enumerated on a FACSCaliber collecting at least 30,000 events. Isotype-matched control antibodies were used to set the limits of background fluorescence.
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2.12. TGF-b and TNF-specific ELISAs For the detection of TGF-b or TNF, a BD biosciences OptEIA™ ELISA kit was used according to the manufacturer’s guidelines. In brief, cytokine-specific capture antibody was coated onto 96-well microtitre plates overnight. Supernatant from BAL was diluted 1:2 in PBS containing 3% FCS and run in triplicate. Cytokine-specific detection antibody conjugated to biotin and avidin-HRP was added to each well simultaneously. Bound antibody was detected with OPD substrate (Sigma), and the reaction was stopped with 2 M sulfuric acid, and absorbance read at 490 nm. The pg/ml of each cytokine was calculated from the standard curve. 2.13. Statistical analysis Statistical significance was evaluated using a two-tailed Student’s t-test, assuming unequal variance. Where more than one comparison was made within an experiment, the Bonferroni correction was applied. 3. Results 3.1. TGF-b prevents pulmonary eosinophilia during C. neoformans infection C57BL/6 mice develop a Th2-driven pulmonary eosinophilia [38], and at the peak of pathogen burden, up to 60%
of airway cells are eosinophils. The role of immunoregulatory cytokines in C. neoformans-induced pulmonary eosinophilia is unclear. We, therefore, examined the effect of TGF-b plasmid DNA delivered nasally 1 week prior to C. neoformans infection. The cellularity of the lung (Fig. 1A) and airways (data not shown) was reduced compared to control plasmid vaccination or administration of PBS. This was most significant 11 days (Fig. 1A) after C. neoformans infection, which corresponded to the peak of pathogen replication. We next assessed the effect of TGF-b plasmid treatment on pulmonary eosinophilia and pathogen burden. A clear association existed between the level of pathogen and eosinophilia (Fig. 1B). Those mice treated with TGF-b had significantly lower pulmonary eosinophils than both control groups but the highest pathogen burden. Both the PBS- and control plasmid-treated group had high eosinophilia but low cfu. IgE is induced during inflammatory Th2 responses and has previously been identified during C. neoformans infection [39]. TGF-b plasmid significantly decreased the level of pathogenspecific IgE in the airways (Fig. 1C) and nasal wash (not shown). The level of C. neoformans-specific IgE in serum was too low to draw any firm conclusions. The CD4+ and CD8+ T cell populations were analyzed by flow cytometry. The total cell numbers for both populations did not significantly differ in the TGF-b plasmid-treated group compared to the control plasmid-treated group (Fig. 1D).
Fig. 1. TGF-b plasmid vaccination prevents pulmonary eosinophilia but inhibits clearance of C. neoformans. TGF-b (closed symbols) or control plasmid (striped symbols) or PBS (open symbols) alone were administered to mice i.n. 1 week prior to C. neoformans infection. Total lung cells were enumerated by trypan blue exclusion 11 days after infection (A). Lung eosinophils were enumerated by flow cytometry and in H&E-stained cytocentrifuge preparations, and the percent (x axis) plotted against the cfu of C. neoformans recovered from the same lung (y axis) (B). C. neoformans-specific IgE was determined in BAL fluid, diluted 1:2 in BSA/PBS, by ELISA. An OD value at 490 nm above 0.2 represented a positive value greater than background levels (measured with uninfected mouse BAL fluid). Results are shown as mean ± S.D. of five mice per group (C). CD4+ and CD8+ T cell subsets in the lung were analyzed by flow cytometry. Total cell numbers did not alter in the TGF-b plasmid-treated group compared to the control plasmid-treated group (D). All results are representative of two to three independent experiments. *P < 0.05 compared to control pCI-neo group.
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3.2. TGF-b prevents lung inflammation but inhibits clearance of influenza virus In the absence of universal vaccine candidates, inhibition of inflammatory T cells presents a novel strategy for therapeutic intervention in the treatment of influenza virus infection. Since the associated pathology is predominantly mediated by Th1 type cytokines, we were interested in comparing the effect of TGF-b during influenza infection with that of C. neoformans infection. TGF-b was administered 1 week prior to intranasal influenza virus infection, and lung compartments were sampled. Mice infected with influenza gradually lose weight, which corresponds to the level of infiltrate into the lung and/or viral load [40]. The reduced T cell infiltrate was accompanied by higher influenza load (P < 0.05) in the lung tissue (Fig. 2A). This was clearly associated with
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increased TGF-b production in those mice treated with the TGF-b plasmid (Fig. 2B). Both control groups (PBS and pCIneo) lost weight until day 4 and then recovered with similar kinetics (Fig. 2C). TGF-b plasmid-treated mice, however, continued to lose weight and were killed at day 7 (Fig. 2C). We have previously shown that a reduction in cellularity or inflammatory cytokine production [40] reduces influenza-induced weight loss, which is due to reduced consolidation in airways. Surprisingly, TGF-b-treated mice also had reduced total numbers (open circles) and percent (closed circles) lymphocytes in the airways (Fig. 2D). Delineating this effect further showed that the percentage (closed circles) of CD4+ (Fig. 2E) but not CD8+ (Fig. 2F) T cells was significantly (P < 0.05) lower in TGF-b−treated mice compared to controls. When total (open circles) T cell subsets were calculated, however, both subsets were reduced (Fig. 2E,F, right axis) (P < 0.05).
Fig. 2. TGF-b prevents lung inflammation but impairs control of influenza virus replication. Influenza virus titre in lung homogenates was determined by plaque assay 7 days post-infection (A) and the results were expressed as mean ± S.D. of five mice. *P < 0.02 compared to control pCI-neo group. This was clearly associated with heightened TGF-b production (B) in TGF-b plasmid-treated mice compared to the PBS- or pCI-neo-treated mice (*P < 0.05). PBS, control plasmid or TGF-b plasmid was administered to mice i.n. 1 week prior to influenza virus infection (C). Mouse weights were monitored for 7 days, and the results were expressed as a percentage of initial starting weights. The percent (closed symbols, left axis) of airway lymphocytes (D), CD4+ T cells (E) and CD8+ T cells (F) was determined by flow cytometry. The actual number of each cell population (open symbols, right axis) was calculated by % lymphocytes gated by flow cytometry × total cell numbers from individual BAL fluid.
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TGF-b plasmid treatment, therefore, affected both the composition of the lymphocyte compartment and the actual magnitude of the lymphocytic response. 3.3. TGF-b modified the expression of both Th1 and Th2 type cytokines TGF-b is able to modify the cytokine phenotype of the immune environment, partly by decreasing IL-12 production by APCs [14]. Therefore, the cytokine profile following influenza virus infection was analyzed by intracellular flow cytometry. The proportion (closed circles) of CD4+ T cells producing IFN-c was reduced (Fig. 3A) but the proportion of IL-4producing cells increased (Fig. 3B) compared to both control groups. However, TGF-b treatment resulted in a decrease (P < 0.05) in both the total number (open circles) of IFN-c and IL-4-producing CD4 + T cells compared to the controls (Fig. 3A, B). A similar decrease in the proportion of IFN-cproducing CD8+ T cells and an increase in the proportion IL-4-producing CD8+ T cells were observed (Fig. 3C, D). In addition, the total number of CD8+ T cells producing either IFN-c or IL-4 decreased in the TGF-b plasmid-treated group (Fig. 3C, D).
ment prior to a primary RSV infection produced similar results in response to the influenza infection model. Lung plaqueforming units were increased (P < 0.05) at day 7 following infection (Fig. 4A). Cellular recruitment to the lung and airways (data not shown) was reduced, however (Fig. 4B). In addition, the total number of CD4+ and CD8+ T cells in the lung also decreased in the TGF-b-treated mice compared to the PBS control (Fig. 4C,D). Interestingly, the proportion of CD4+ (Fig. 4E) and CD8+ (Fig. 4F) T cells in the mediastinal lymph node (MLN) increased in the TGF-b−treated group. Further analysis showed that activated CD4+ and CD8+ also decreased (Fig. 4G,H), measured as the CD45RBlo populations. Similar effects on the MLNs were also observed in the influenza virus infection model (data not shown). The reduced cellularity in the lung was not due to impaired antigen endocytosis by resident alveolar or lung APCs, since the uptake of FITC-labeled dextran was similar regardless of TGF-b treatment (Fig. 5A). It is interesting to note that the majority of CD11b+ cells in the airways contained FITCdextran from 4 days onwards, whereas only low expression was observed in CD11b+ lung interstitium cells (Fig. 5A). Reduced cellularity may, however, be explained by a reduction in inflammatory signals, in particular TNF (Fig. 5B), in the lung microenvironment.
3.4. TGF-b treatment reduces pulmonary inflammation but impairs clearance of RSV 4. Discussion So far, we have demonstrated that TGF-b plasmid prevents pulmonary inflammation in response to both C. neoformans and influenza virus. Intranasal TGF-b plasmid treat-
In this report, we demonstrate that nasal administration of plasmid DNA encoding active TGF-b suppresses T cell
Fig. 3. TGF-b affects both Th1 and Th2 type cytokines. PBS, control plasmid or TGF-b plasmid was administered to mice i.n. 1 week prior to influenza virus infection. Seven days after infection, a BAL fluid from the airways was centrifuged, and the cells were stained with antibodies to CD4 and either IFN-c (A), or IL-4 (B), or CD8 and either IFN-c (C) or IL-4 (D). The percent (closed symbols, left axis) cells expressing these markers were determined by flow cytometry. The total number of cells expressing each marker was calculated as follows: % cells expressing marker × total lymphocytes (open symbols, right axis). Each point represents an individual mouse and is representative of two to three separate experiments.
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Fig. 4. TGF-b prevents lung inflammation but inhibits clearance of RSV. PBS (open symbols), control plasmid (striped symbols) or TGF-b plasmid (closed symbols) was administered to mice i.n. 1 week prior to RSV infection. Seven days after infection, the number of pfu in whole lung homogenate was determined by plaque assay (A). The total number of cells in the lung was counted by trypan blue exclusion (B). Lung cells were stained with anti-CD4 (C) or anti-CD8 (D) antibodies and analyzed by flow cytometry. MLNs were also removed and single cell suspensions stained with antibodies to CD4 (E), CD8 (F), CD4/CD45RB (G) or CD8/CD45RB (H). The percent cells expressing these markers was determined by flow cytometry. Total numbers were calculated by % positive cells × total lymphocytes. Each point represents an individual mouse and is representative of two to three separate experiments. *P < 0.05 compared to control pCI-neo group.
responses in the airway and sub-mucosal tissue during both Th1 and Th2 immune pathology elicited by viral and nonviral respiratory infection. It is clear that in some animal models and certain individuals, C. neoformans drives a pulmonary eosinophilic response [38]. In the C57BL/6 mouse model of C. neoformans infection, it has previously been shown that CD4+ T cells and IL-5 are critical for pulmonary eosinophilia [41]. In the current study, we show that TGF-b abrogates cell infiltration into the lung (including eosinophils) but at the same time, allows the pathogen to replicate unchecked, therefore, supporting the concept that eosinophilia is a T celldriven immune pathology [41].
The reduction of eosinophilic pathology is interesting, since TGF-b (in combination with IL-13) has been shown to increase eotaxin expression [42], and previous studies imply that its anti-inflammatory properties are more prominent in Th1 conditions [43,44]. A critical factor contributing to the effect of TGF-b is the site of administration of the plasmid. The lung airways have a unique cellular composition comprised almost entirely of alveolar macrophages, which themselves are thought to be immune suppressive [45–47]. This is unlike that exhibited at other mucosal sites. The results also show that a reduction in one T helper cell population is not compensated for by the reciprocal popula-
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Fig. 5. TGF-b does not influence endocytosis but decreases TNF production. FITC-dextran was administered i.n. to TGF-b-treated or control mice 1 day prior to influenza virus infection (A). Lung (top panels) and airway (bottom panels) cells were sampled at various time points (only the results for day 4 are shown). Recovered cells were stained with PE-conjugated antibodies to CD11b and the co-expression of FITC and PE examined by flow cytometry. The plots are representative of two independent experiments containing four mice per group. TGF-b decreases (*P < 0.05) the production of TNF in the airways (B) as measured by cytokine-specific ELISA, compared to both PBS and pCI-neo treatment.
tion. Even in severely eosinophilic situations, both Th1 and Th2 cells are reduced in number. Similarly, TGF-b is reported to induce aE mRNA expression, which with b7 forms an integrin (aEb7 or CD103) critical for homing T cells to the mucosa and especially the intra-epithelial compartment [48,49]. The reduced cellularity in the lung and the lack of an effect on CD103 expression (data not shown) rules out this function in the current study. Delineating the effect of TGF-b1 is confusing, since it plays both a pro-inflammatory and anti-inflammatory role, depending on the stage of the immune response. Its pro-inflammatory influences include promotion of T cell migration [50], inhibition of activation-induced cell death [51] and differentiation of monocytes into dendritic cells [52]. Anti-inflammatory actions are required to prevent prolonged inflammation that may be harmful to the host. Both the pro- and antiinflammatory actions can also lead to pathology. TGFb1 induces the release of other inflammatory molecules from fibroblasts taken from the joints of patients with rheumatoid arthritis [53], and in an animal model of rheumatoid arthritis, it exacerbates the inflammatory response [54]. Defective TGFb1 production has been implicated in a variety of inflammatory conditions including Crohn’s disease, multiple sclerosis
and rheumatoid arthritis [55]. In the current study, we only observed anti-inflammatory actions and reduced cell activation and cell numbers. The role of TGF-b1 in migration is similarly confusing. It inhibits the activation of rat endothelial cells, including the adhesion and migration of polymorphonuclear leukocytes, platelets and lymphocytes [56]. The increased risk of postoperative infections in patients receiving transfusions has been linked to the presence of TGF-b1 and subsequent reduced neutrophil migration [57]. Recent studies suggest that its effect on migration will depend on the cytokine polarity of the circulating T cell. Th2 cells are reported to express CCR3 and CCR4, whereas Th1 and Th0 cells express CCR5 and CXCR3. TGFb1 induces the expression of CCR4 and CCR7 effectively targeting the T cell to secondary lymph nodes [58]. This may explain why TGF-b1 treatment in our study resulted in the increase in T cells in the lung-associated MLN, which may reflect a reduction in migration. It is probably significant that we administered plasmid TGF-b before the lung pathogen, since it has been shown to affect naïve but not memory T cells [59]. Although newly differentiated Th1 and Th2 cells express TGF-bRII, mature T cells down-regulate it [60]. This receptor is the only one
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that binds free TGF-b [61]. Infection of the lung is often accompanied by exaggerated inflammation, most of which is non-antigen specific. Even during a re-infection, naïve cells traffic into the lung with memory T cell populations. TGF-b plasmid treatment may, therefore, be beneficial in established disease. Overall, administration of plasmid TGF-b DNA by the intranasal route increases unactivated T cells in the lung draining MLNs. This may reflect a reduction in the capacity of lung APCs to process and present pathogen-derived antigens to T cells in the node. Reduced presentation of antigen to T cells would explain the reduced activation of T cells. However, we did not observe any effect on the ability of APCs to endocytose FITC-dextran. Alternatively, reduced T cell activation may result in tolerance induction or low responsiveness. Indeed, a reduction in TNF production in the airways following TGF-b treatment is consistent with diminished T cell activation. TGF-b is vital for T cell homeostasis. If the TGF-b type II receptor is rendered non-functional, T cells become spontaneously activated, leading to multiple inflammatory foci [62]. In the current study, the reduction in lung T cells is likely to be caused by a number of factors. A single administration of plasmid DNA encoding TGF-b reduces the excessive inflammatory response to a variety of lung pathogens. Such a strategy could be used to prevent excessive inflammation but may also have the unwanted result of an increase in replicating pathogen and associated disease. Further studies or modified approaches need to be undertaken to fully appreciate the potential of therapeutic TGF-b treatment.
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Acknowledgements We wish to acknowledge Barry Rouse, University of Tennessee, USA, for the provision of the plasmid DNA. We wish to acknowledge the National Asthma Campaign, UK for their financial support (grant number 00/007).
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Original article
TGF-b prevents eosinophilic lung disease but impairs pathogen clearance Andrew Evan Williams a,*,1, Ian Robert Humphreys a,*,1, Megan Cornere a, Lorna Edwards a, Aaron Rae b, Tracy Hussell b a
Kennedy Institute of Rheumatology, Charing Cross Campus, Imperial College London, London W6 8LM, UK b Centre for Molecular Microbiology and Infection, Department of Biological Sciences, South Kensington, Imperial College London, London SW7 2AZ, UK Received 5 August 2004; accepted 10 November 2004 Available online 19 March 2005
Abstract Respiratory infections are the third leading cause of death worldwide. Complications arise directly as a consequence of pathogen replication or indirectly due to aberrant or excessive immune responses. In the following report, we evaluate the efficacy, in a murine model, of nasally delivered DNA encoding TGF-b1 to suppress immunopathology in response to a variety of infectious agents. A single nasal administration suppressed lymphocyte responses to Cryptococcus neoformans, influenza virus and respiratory syncytial virus. The suppression did not depend on the phenotype of the responding T cell, since both Th1 and Th2 responses were affected. During Th2-inducing infection, pulmonary eosinophilic responses were significantly suppressed. In all cases, however, suppressed immunity correlated with increased susceptibility to infection. We conclude that nasal TGF-b treatment could be used to prevent pulmonary, pathogen-driven eosinophilic disease, although anti-pathogen strategies will need to be administered concordantly. © 2005 Elsevier SAS. All rights reserved. Keywords: Viral; Cytokines; Inflammation; Lung; Mouse
1. Introduction TGF-b plays a central role in the regulation of inflammatory responses including cell proliferation, extracellular matrix deposition, cell migration and differentiation [1]. It is regarded as an immunosuppressive factor and can down-regulate both Th1 and Th2 type responses in addition to its profibrotic action [2–4]. Three isoforms exist in mammals (TGF-beta 1, -beta 2 and -beta 3), each isoform being encoded on different chromosomes [4]. TGF-b is produced by most cell types and is secreted in a latent form, bound to latency-associated peptide (LAP), requiring dissolution for biological activity [5,6]. The activation of latent TGF-b is a crucial regulatory step in con-
Abbreviations: APC, antigen-presenting cell; BAL, broncho-alveolar lavage; IFN-c, interferon-c; IL-4, interleukin-4; RSV, respiratory syncytial virus; TGF-b, transforming growth factor-b; Th, T helper; TNF, tumour necrosis factor. * Corresponding authors. Tel.: +44 20 8383 4769; fax: +44 20 8383 4499. E-mail address: [email protected] (A.E. Williams). 1 These authors contributed equally to this work. 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.11.012
trolling inflammation. LAP may re-associate with TGF-b, rendering it inactive [5]. The 45-kDa protein, decorin, is also expressed in pulmonary tissues and may play a role in counteracting the profibrotic actions of TGF-b [7]. The role of TGF-b is clearly identified in knockout mice, which generate uncontrolled inflammatory responses in several organs. Production of TGF-b is thought to mediate oral tolerance [8,9] and administration of recombinant TGF-b1 suppresses encephalomyelitis, inflammatory bowel disease and uveitis [8,10–12]. However, such therapy requires repeated administration of substantial quantities of protein [10]. TGF-b can suppress both Th1 and Th2 cells via cell cycle inhibition, and it acts to directly antagonize interleukin-4 (IL-4) and IL-12 signaling pathways [13,14]. Several studies report a deviation of T cells towards a Th2 phenotype under the influence of TGF-b. Immune privilege in the eye is mediated by TGF-b and leads to Th2 cytokine predominance [15]. TGF-b plays a critical role in mucosal immune homeostasis; preventing detrimental inflammatory responses, while promoting non-inflammatory dimeric IgA. However, this balance is affected by the presence of an infection that requires
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the induction of an immune response for eradication. Resistance of certain mouse strains to visceral leishmaniasis [16,17] and infection with Plasmodium species [17] depends on low production of TGF-b and high levels of interferon-c (IFN-c). Recombinant TGF-b enhances the survival of Salmonella typhimurium in the mouse gut [18], presumably by downregulating inflammation. However, the role of TGF-b at other mucosal sites is not well understood. Its primary role in the immunopathogenesis of the advanced forms of pulmonary tuberculosis is clear [19]. Higher levels of TGF-b are observed in pulmonary macrophages [20], tuberculosis pleurisy [21] and during in vivo infection [19] possibly via engagement of Toll-like receptor 2 [22]. However, its importance for other pulmonary infections is not well understood. In the following report, we determine that overexpression of TGF-b, through the administration of plasmid DNA expressing the gene for TGF-b1, in a variety of lung infection models reduces inflammatory diseases. However, the ability to clear the pathogen was reduced, and hence, illness was enhanced. Three different infection models were tested so as to include both viral and non-viral disease and Th1-versus Th2-driven immunopathology. Influenza virus is a causative agent of both epidemic and pandemic respiratory viral illness, with over 26,000 deaths attributed to the last outbreak in Britain alone [23]. Though pivotal for viral clearance, overexuberant, Th1-driven T cell responses result in airway occlusion [24,25] and contribute significantly to pathology [26]. Infection of BALB/c mice with influenza reflects many of the features of the human disease, with cachexia, weight loss, fever and pulmonary occlusion. Respiratory syncytial virus (RSV) is another significant respiratory pathogen that causes bronchiolitis in infants [27]. Children hospitalized with bronchiolitis often suffer recurrent wheezing and are frequently diagnosed as asthmatic [28]. There is strong evidence that viral bronchiolitis is a T cell-mediated immunopathological condition [29,30]. In the BALB/c mouse, primary intranasal infection with RSV causes mild self-limiting illness characterized by Th1-driven T cell infiltration. The fungus Cryptococcus neoformans is a significant respiratory pathogen causing pulmonary eosinophilia in immunocompetent hosts [31] and summer-type hypersensitivity pneumonitis in Japan [32]. In immune-deficient patients, dissemination of the pathogen occurs due to the inability of the host to limit the infection [33,34]. The C57BL/6 model of C. neoformans infection mimics the situation in humans and is characterized by Th2driven pulmonary eosinophilia. Lung administration of a DNA plasmid provided a useful method of expressing an active form of TGF-b1. TGF-b1 has multiple effects on a variety of inflammatory cells. The results demonstrate that TGF-b moderates both Th2-driven eosinophilic and Th1 cell-mediated pathology but that this antiinflammatory treatment allows unchecked pathogen replication. We conclude that TGF-b treatment is a viable strategy for preventing pulmonary inflammation but anti-pathogen therapy will need to be administered concurrently.
2. Materials and methods 2.1. Mice and pathogen stocks Eight- to 10-week-old female BALB/c and C57BL/6 mice (Harlan Olac Ltd., Bicester, UK) were kept in specifiedpathogen-free conditions, according to institutional and Home Office (UK) guidelines. Influenza A strain X31 (haemagglutinin [HA] titre 1024) was a kind gift from Dr. Alan Douglas (National Institute for Medical Research, London, UK). RSV was grown in HEp-2 cells and assayed for infectivity as previously described [35]. All stocks were free of mycoplasma, assayed by DNA hybridization (Gen-Probe Inc., San Diego, CA). C. neoformans 52 was obtained from the American Type Culture Collection (no. 24067, Rockville, MD, USA). For infection, yeast was grown to stationary phase (48–72 h) at room temperature on a shaker in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, USA). The cultures were washed in saline, counted on a haemocytometer and diluted in sterile non-pyrogenic saline to the required infective dose. The yeast were plated on Sabouraud agar plates (Difco), and colonies were counted after 48 h culture at room temperature to assess viability. 2.2. Plasmid preparation Plasmid DNA, either pCI-neo (control plasmid with no insert) or pCI-neo containing the gene for murine TGF-b1 was a kind gift from Barry Rouse (Tennessee, USA). DH5a competent cells (Invitrogen, UK) were transformed with plasmid DNA under ampicillin selection. Experimental quantities of DNA were attained using endotoxin-free giga preparation (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The integrity of the plasmid was analyzed on a 0.6% agarose gel in the presence of ethidium bromide. Expression levels of TGF-b1 in murine lung tissue, following a single administration, have been tested previously [36]. Expression of TGF-b from plasmid DNA was assayed by transfection of COS-7 cells, followed by specific immunohistochemistry and TGF-b specific ELISA (Becton Dickinson). 2.3. Experimental procedure Mice were administered either 50 µl PBS or 100 µg of either control or TGF-b-expressing plasmid DNA in 50 µl PBS intranasally (i.n.), 1 week prior to i.n. infection with 103 cfu C. neoformans, 106 pfu RSV or 50 HA units of influenza virus. C. neoformans infections, and RSV and influenza infections were performed in C57BL/6 and BALB/c mice, respectively. Mice were monitored daily, and weight loss was measured throughout infection. Mice were killed at various time points by the injection of 3 mg pentobarbitone and exsanguination of the femoral vessels. 2.4. Cell recovery Broncho-alveolar lavage (BAL) fluid, lung tissue and serum were obtained from each mouse as described previ-
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ously [37]. In brief, lungs were inflated six times with 1.5 ml of Eagle’s minimum essential medium (Sigma) containing 10 mM EDTA and kept on ice. One-hundred milliliters of this BAL fluid was cytocentrifuged onto glass slides, fixed in methanol and stained with haematoxylin and eosin (H and E; Sigma, Dorset UK). The remainder was centrifuged, the supernatant decanted, and the cell pellet re-suspended to 1 ×106 cells per ml in RPMI (Sigma) containing 10% fetal calf serum (FCS), 2 mM/ml L -glutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin (R10F). Lung tissue was homogenized to obtain single cell suspensions, the red blood cells were lysed and the cell pellet re-suspended at 1 × 106 cells per ml in R10F. 2.5. Enumeration of eosinophils Granulocytes were enumerated by flow cytometry based on their size (forward scatter) and granularity (side scatter). From cytopsin preparations, the proportion of eosinophils was determined by their distinctive nuclear morphology and the presence of acidophilic red granules. Neutrophils were identified by the presence of multi-lobed nuclei and the absence of acidophilic granules. 2.6. Flow cytometry Cells (1 × 106) obtained from the airways or the lungs were stained with the following antibody combinations. (1) AntiCD45RB FITC, anti-CD4 allophycocyanin (APC) and antiCD8 PerCP (all BD Pharmingen, Heidelberg, Germany). (2) To detect intracellular cytokines, 1 × 106 cells were incubated with 50 ng/ml PMA (Sigma-Aldrich), 500 ng ionomycin (Calbiochem, Nottingham, UK) and 10 µg/ml brefeldin A (Sigma) for 4 h at 37 °C. Cells were stained with antiCD4 APC and anti-CD8 PerCP on ice for 30 min, washed and then fixed in 2% formaldehyde for 20 min at room temperature. Cells were permeabilized with 0.5% saponin in PBS containing 1% BSA and 0.1% azide for 10 min. A combination of anti-IFNc FITC and anti-IL-4 PE antibodies (BD Pharmingen), diluted in saponin buffer, was then added to the cells. After 30 min, cells were washed once in saponin buffer and twice in PBS containing 0.1% azide and 1% BSA. Samples were analyzed on a FACSCaliber (BD Biosciences, Belgium), collecting data on at least 30,000 lymphocytes. 2.7. Influenza plaque assays Clearance of influenza was assessed from lung homogenates following virus challenge. Homogenized cells were freeze-thawed three times, centrifuged at 4000 × g, and supernatants titrated in doubling dilutions on Madin–Darby canine kidney cell monolayers in flat-bottomed 96-well plates. After incubation at room temperature for 3 h, samples were overlayed with methycellulose and incubated for 72 h at 37 °C. Cell monolayers were washed and incubated with antiinfluenza antibody (Serotec UK, Oxford, UK), followed by
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anti-mouse-HRP (Dako Ltd., Cambridge, UK), and infected cells were detected using 3-amino-9-ethylcarbazole (AEC, Sigma). Infectious units were enumerated by light microscopy, and total plaque-forming units per lung were quantified (number of plaques _ dilution factor _ total volume of lung homogenate). 2.8. RSV plaque assay Infectivity was determined in lung homogenates by infection of HEp-2 cell monolayers for 2 h at 37 °C in serum-free RPMI, prior to overlaying with 150 µl R10F. After 48 h, the monolayer was washed in PBS with 1% BSA and fixed with 100 µl per well of methanol and 0.6% H2O2 for 20 min. Cells were then stained for anti-RSV-HRP (Biogenesis, Poole, Dorset, UK) for 1 h, washed twice with BSA/PBS, and plaques visualized by addition of AEC substrate for 30 min. Plaques were enumerated by light microscopy. 2.9. Enumeration of C. neoformans burden Lungs were homogenized by passage through 100-µm cell strainers. Homogenate (100 µl) was diluted in sterile PBS and incubated at room temperature for 48 h on Sabouraud dextrose agar plates (Sigma). The total colony-forming units per lung was then determined (number of colonies × dilution factor × original cell suspension volume). 2.10. C. neoformans specific IgE Heat-killed C. neoformans (2 × 105 cfu/ml) in PBS was used to coat 96-well micro-titre plates overnight at room temperature on a shaker. After blocking with 3% BSA/PBS for 2 h at room temperature, BAL fluid diluted 1:2 in BSA/PBS was added for a further hour at room temperature. Bound IgE antibody was detected with anti-IgE-HRP (Serotec UK) in BSA/PBS. Plates were washed again, and bound antibody detected with o-phenylenediamine dihydrochloride (OPD, Sigma) substrate. The reaction was stopped with 2 M sulfuric acid. Known IgE standard concentration was unavailable, therefore, differences between treatment groups were measured by absorbance at 490 nm, and background measured as a value of 0.2 using uninfected mouse BAL fluid. 2.11. Analysis of FITC-dextran uptake BALB/c mice were i.n. administered 100 µg of plasmid DNA (TGF-b or control) or PBS in 50-µl volume, twice at weekly intervals. Seven days later, mice were i.n. infected with 50 HA units of influenza virus. One day later, 50 µg of FITC-dextran in 50 µl was administered i.n. At various time points, lung, BAL and mediatinal lymph nodes were sampled for flow cytometry. Cells expressing FITC and anti-CD11b PE (BD Pharmingen) were enumerated on a FACSCaliber collecting at least 30,000 events. Isotype-matched control antibodies were used to set the limits of background fluorescence.
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2.12. TGF-b and TNF-specific ELISAs For the detection of TGF-b or TNF, a BD biosciences OptEIA™ ELISA kit was used according to the manufacturer’s guidelines. In brief, cytokine-specific capture antibody was coated onto 96-well microtitre plates overnight. Supernatant from BAL was diluted 1:2 in PBS containing 3% FCS and run in triplicate. Cytokine-specific detection antibody conjugated to biotin and avidin-HRP was added to each well simultaneously. Bound antibody was detected with OPD substrate (Sigma), and the reaction was stopped with 2 M sulfuric acid, and absorbance read at 490 nm. The pg/ml of each cytokine was calculated from the standard curve. 2.13. Statistical analysis Statistical significance was evaluated using a two-tailed Student’s t-test, assuming unequal variance. Where more than one comparison was made within an experiment, the Bonferroni correction was applied. 3. Results 3.1. TGF-b prevents pulmonary eosinophilia during C. neoformans infection C57BL/6 mice develop a Th2-driven pulmonary eosinophilia [38], and at the peak of pathogen burden, up to 60%
of airway cells are eosinophils. The role of immunoregulatory cytokines in C. neoformans-induced pulmonary eosinophilia is unclear. We, therefore, examined the effect of TGF-b plasmid DNA delivered nasally 1 week prior to C. neoformans infection. The cellularity of the lung (Fig. 1A) and airways (data not shown) was reduced compared to control plasmid vaccination or administration of PBS. This was most significant 11 days (Fig. 1A) after C. neoformans infection, which corresponded to the peak of pathogen replication. We next assessed the effect of TGF-b plasmid treatment on pulmonary eosinophilia and pathogen burden. A clear association existed between the level of pathogen and eosinophilia (Fig. 1B). Those mice treated with TGF-b had significantly lower pulmonary eosinophils than both control groups but the highest pathogen burden. Both the PBS- and control plasmid-treated group had high eosinophilia but low cfu. IgE is induced during inflammatory Th2 responses and has previously been identified during C. neoformans infection [39]. TGF-b plasmid significantly decreased the level of pathogenspecific IgE in the airways (Fig. 1C) and nasal wash (not shown). The level of C. neoformans-specific IgE in serum was too low to draw any firm conclusions. The CD4+ and CD8+ T cell populations were analyzed by flow cytometry. The total cell numbers for both populations did not significantly differ in the TGF-b plasmid-treated group compared to the control plasmid-treated group (Fig. 1D).
Fig. 1. TGF-b plasmid vaccination prevents pulmonary eosinophilia but inhibits clearance of C. neoformans. TGF-b (closed symbols) or control plasmid (striped symbols) or PBS (open symbols) alone were administered to mice i.n. 1 week prior to C. neoformans infection. Total lung cells were enumerated by trypan blue exclusion 11 days after infection (A). Lung eosinophils were enumerated by flow cytometry and in H&E-stained cytocentrifuge preparations, and the percent (x axis) plotted against the cfu of C. neoformans recovered from the same lung (y axis) (B). C. neoformans-specific IgE was determined in BAL fluid, diluted 1:2 in BSA/PBS, by ELISA. An OD value at 490 nm above 0.2 represented a positive value greater than background levels (measured with uninfected mouse BAL fluid). Results are shown as mean ± S.D. of five mice per group (C). CD4+ and CD8+ T cell subsets in the lung were analyzed by flow cytometry. Total cell numbers did not alter in the TGF-b plasmid-treated group compared to the control plasmid-treated group (D). All results are representative of two to three independent experiments. *P < 0.05 compared to control pCI-neo group.
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3.2. TGF-b prevents lung inflammation but inhibits clearance of influenza virus In the absence of universal vaccine candidates, inhibition of inflammatory T cells presents a novel strategy for therapeutic intervention in the treatment of influenza virus infection. Since the associated pathology is predominantly mediated by Th1 type cytokines, we were interested in comparing the effect of TGF-b during influenza infection with that of C. neoformans infection. TGF-b was administered 1 week prior to intranasal influenza virus infection, and lung compartments were sampled. Mice infected with influenza gradually lose weight, which corresponds to the level of infiltrate into the lung and/or viral load [40]. The reduced T cell infiltrate was accompanied by higher influenza load (P < 0.05) in the lung tissue (Fig. 2A). This was clearly associated with
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increased TGF-b production in those mice treated with the TGF-b plasmid (Fig. 2B). Both control groups (PBS and pCIneo) lost weight until day 4 and then recovered with similar kinetics (Fig. 2C). TGF-b plasmid-treated mice, however, continued to lose weight and were killed at day 7 (Fig. 2C). We have previously shown that a reduction in cellularity or inflammatory cytokine production [40] reduces influenza-induced weight loss, which is due to reduced consolidation in airways. Surprisingly, TGF-b-treated mice also had reduced total numbers (open circles) and percent (closed circles) lymphocytes in the airways (Fig. 2D). Delineating this effect further showed that the percentage (closed circles) of CD4+ (Fig. 2E) but not CD8+ (Fig. 2F) T cells was significantly (P < 0.05) lower in TGF-b−treated mice compared to controls. When total (open circles) T cell subsets were calculated, however, both subsets were reduced (Fig. 2E,F, right axis) (P < 0.05).
Fig. 2. TGF-b prevents lung inflammation but impairs control of influenza virus replication. Influenza virus titre in lung homogenates was determined by plaque assay 7 days post-infection (A) and the results were expressed as mean ± S.D. of five mice. *P < 0.02 compared to control pCI-neo group. This was clearly associated with heightened TGF-b production (B) in TGF-b plasmid-treated mice compared to the PBS- or pCI-neo-treated mice (*P < 0.05). PBS, control plasmid or TGF-b plasmid was administered to mice i.n. 1 week prior to influenza virus infection (C). Mouse weights were monitored for 7 days, and the results were expressed as a percentage of initial starting weights. The percent (closed symbols, left axis) of airway lymphocytes (D), CD4+ T cells (E) and CD8+ T cells (F) was determined by flow cytometry. The actual number of each cell population (open symbols, right axis) was calculated by % lymphocytes gated by flow cytometry × total cell numbers from individual BAL fluid.
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TGF-b plasmid treatment, therefore, affected both the composition of the lymphocyte compartment and the actual magnitude of the lymphocytic response. 3.3. TGF-b modified the expression of both Th1 and Th2 type cytokines TGF-b is able to modify the cytokine phenotype of the immune environment, partly by decreasing IL-12 production by APCs [14]. Therefore, the cytokine profile following influenza virus infection was analyzed by intracellular flow cytometry. The proportion (closed circles) of CD4+ T cells producing IFN-c was reduced (Fig. 3A) but the proportion of IL-4producing cells increased (Fig. 3B) compared to both control groups. However, TGF-b treatment resulted in a decrease (P < 0.05) in both the total number (open circles) of IFN-c and IL-4-producing CD4 + T cells compared to the controls (Fig. 3A, B). A similar decrease in the proportion of IFN-cproducing CD8+ T cells and an increase in the proportion IL-4-producing CD8+ T cells were observed (Fig. 3C, D). In addition, the total number of CD8+ T cells producing either IFN-c or IL-4 decreased in the TGF-b plasmid-treated group (Fig. 3C, D).
ment prior to a primary RSV infection produced similar results in response to the influenza infection model. Lung plaqueforming units were increased (P < 0.05) at day 7 following infection (Fig. 4A). Cellular recruitment to the lung and airways (data not shown) was reduced, however (Fig. 4B). In addition, the total number of CD4+ and CD8+ T cells in the lung also decreased in the TGF-b-treated mice compared to the PBS control (Fig. 4C,D). Interestingly, the proportion of CD4+ (Fig. 4E) and CD8+ (Fig. 4F) T cells in the mediastinal lymph node (MLN) increased in the TGF-b−treated group. Further analysis showed that activated CD4+ and CD8+ also decreased (Fig. 4G,H), measured as the CD45RBlo populations. Similar effects on the MLNs were also observed in the influenza virus infection model (data not shown). The reduced cellularity in the lung was not due to impaired antigen endocytosis by resident alveolar or lung APCs, since the uptake of FITC-labeled dextran was similar regardless of TGF-b treatment (Fig. 5A). It is interesting to note that the majority of CD11b+ cells in the airways contained FITCdextran from 4 days onwards, whereas only low expression was observed in CD11b+ lung interstitium cells (Fig. 5A). Reduced cellularity may, however, be explained by a reduction in inflammatory signals, in particular TNF (Fig. 5B), in the lung microenvironment.
3.4. TGF-b treatment reduces pulmonary inflammation but impairs clearance of RSV 4. Discussion So far, we have demonstrated that TGF-b plasmid prevents pulmonary inflammation in response to both C. neoformans and influenza virus. Intranasal TGF-b plasmid treat-
In this report, we demonstrate that nasal administration of plasmid DNA encoding active TGF-b suppresses T cell
Fig. 3. TGF-b affects both Th1 and Th2 type cytokines. PBS, control plasmid or TGF-b plasmid was administered to mice i.n. 1 week prior to influenza virus infection. Seven days after infection, a BAL fluid from the airways was centrifuged, and the cells were stained with antibodies to CD4 and either IFN-c (A), or IL-4 (B), or CD8 and either IFN-c (C) or IL-4 (D). The percent (closed symbols, left axis) cells expressing these markers were determined by flow cytometry. The total number of cells expressing each marker was calculated as follows: % cells expressing marker × total lymphocytes (open symbols, right axis). Each point represents an individual mouse and is representative of two to three separate experiments.
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Fig. 4. TGF-b prevents lung inflammation but inhibits clearance of RSV. PBS (open symbols), control plasmid (striped symbols) or TGF-b plasmid (closed symbols) was administered to mice i.n. 1 week prior to RSV infection. Seven days after infection, the number of pfu in whole lung homogenate was determined by plaque assay (A). The total number of cells in the lung was counted by trypan blue exclusion (B). Lung cells were stained with anti-CD4 (C) or anti-CD8 (D) antibodies and analyzed by flow cytometry. MLNs were also removed and single cell suspensions stained with antibodies to CD4 (E), CD8 (F), CD4/CD45RB (G) or CD8/CD45RB (H). The percent cells expressing these markers was determined by flow cytometry. Total numbers were calculated by % positive cells × total lymphocytes. Each point represents an individual mouse and is representative of two to three separate experiments. *P < 0.05 compared to control pCI-neo group.
responses in the airway and sub-mucosal tissue during both Th1 and Th2 immune pathology elicited by viral and nonviral respiratory infection. It is clear that in some animal models and certain individuals, C. neoformans drives a pulmonary eosinophilic response [38]. In the C57BL/6 mouse model of C. neoformans infection, it has previously been shown that CD4+ T cells and IL-5 are critical for pulmonary eosinophilia [41]. In the current study, we show that TGF-b abrogates cell infiltration into the lung (including eosinophils) but at the same time, allows the pathogen to replicate unchecked, therefore, supporting the concept that eosinophilia is a T celldriven immune pathology [41].
The reduction of eosinophilic pathology is interesting, since TGF-b (in combination with IL-13) has been shown to increase eotaxin expression [42], and previous studies imply that its anti-inflammatory properties are more prominent in Th1 conditions [43,44]. A critical factor contributing to the effect of TGF-b is the site of administration of the plasmid. The lung airways have a unique cellular composition comprised almost entirely of alveolar macrophages, which themselves are thought to be immune suppressive [45–47]. This is unlike that exhibited at other mucosal sites. The results also show that a reduction in one T helper cell population is not compensated for by the reciprocal popula-
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Fig. 5. TGF-b does not influence endocytosis but decreases TNF production. FITC-dextran was administered i.n. to TGF-b-treated or control mice 1 day prior to influenza virus infection (A). Lung (top panels) and airway (bottom panels) cells were sampled at various time points (only the results for day 4 are shown). Recovered cells were stained with PE-conjugated antibodies to CD11b and the co-expression of FITC and PE examined by flow cytometry. The plots are representative of two independent experiments containing four mice per group. TGF-b decreases (*P < 0.05) the production of TNF in the airways (B) as measured by cytokine-specific ELISA, compared to both PBS and pCI-neo treatment.
tion. Even in severely eosinophilic situations, both Th1 and Th2 cells are reduced in number. Similarly, TGF-b is reported to induce aE mRNA expression, which with b7 forms an integrin (aEb7 or CD103) critical for homing T cells to the mucosa and especially the intra-epithelial compartment [48,49]. The reduced cellularity in the lung and the lack of an effect on CD103 expression (data not shown) rules out this function in the current study. Delineating the effect of TGF-b1 is confusing, since it plays both a pro-inflammatory and anti-inflammatory role, depending on the stage of the immune response. Its pro-inflammatory influences include promotion of T cell migration [50], inhibition of activation-induced cell death [51] and differentiation of monocytes into dendritic cells [52]. Anti-inflammatory actions are required to prevent prolonged inflammation that may be harmful to the host. Both the pro- and antiinflammatory actions can also lead to pathology. TGFb1 induces the release of other inflammatory molecules from fibroblasts taken from the joints of patients with rheumatoid arthritis [53], and in an animal model of rheumatoid arthritis, it exacerbates the inflammatory response [54]. Defective TGFb1 production has been implicated in a variety of inflammatory conditions including Crohn’s disease, multiple sclerosis
and rheumatoid arthritis [55]. In the current study, we only observed anti-inflammatory actions and reduced cell activation and cell numbers. The role of TGF-b1 in migration is similarly confusing. It inhibits the activation of rat endothelial cells, including the adhesion and migration of polymorphonuclear leukocytes, platelets and lymphocytes [56]. The increased risk of postoperative infections in patients receiving transfusions has been linked to the presence of TGF-b1 and subsequent reduced neutrophil migration [57]. Recent studies suggest that its effect on migration will depend on the cytokine polarity of the circulating T cell. Th2 cells are reported to express CCR3 and CCR4, whereas Th1 and Th0 cells express CCR5 and CXCR3. TGFb1 induces the expression of CCR4 and CCR7 effectively targeting the T cell to secondary lymph nodes [58]. This may explain why TGF-b1 treatment in our study resulted in the increase in T cells in the lung-associated MLN, which may reflect a reduction in migration. It is probably significant that we administered plasmid TGF-b before the lung pathogen, since it has been shown to affect naïve but not memory T cells [59]. Although newly differentiated Th1 and Th2 cells express TGF-bRII, mature T cells down-regulate it [60]. This receptor is the only one
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that binds free TGF-b [61]. Infection of the lung is often accompanied by exaggerated inflammation, most of which is non-antigen specific. Even during a re-infection, naïve cells traffic into the lung with memory T cell populations. TGF-b plasmid treatment may, therefore, be beneficial in established disease. Overall, administration of plasmid TGF-b DNA by the intranasal route increases unactivated T cells in the lung draining MLNs. This may reflect a reduction in the capacity of lung APCs to process and present pathogen-derived antigens to T cells in the node. Reduced presentation of antigen to T cells would explain the reduced activation of T cells. However, we did not observe any effect on the ability of APCs to endocytose FITC-dextran. Alternatively, reduced T cell activation may result in tolerance induction or low responsiveness. Indeed, a reduction in TNF production in the airways following TGF-b treatment is consistent with diminished T cell activation. TGF-b is vital for T cell homeostasis. If the TGF-b type II receptor is rendered non-functional, T cells become spontaneously activated, leading to multiple inflammatory foci [62]. In the current study, the reduction in lung T cells is likely to be caused by a number of factors. A single administration of plasmid DNA encoding TGF-b reduces the excessive inflammatory response to a variety of lung pathogens. Such a strategy could be used to prevent excessive inflammation but may also have the unwanted result of an increase in replicating pathogen and associated disease. Further studies or modified approaches need to be undertaken to fully appreciate the potential of therapeutic TGF-b treatment.
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Acknowledgements We wish to acknowledge Barry Rouse, University of Tennessee, USA, for the provision of the plasmid DNA. We wish to acknowledge the National Asthma Campaign, UK for their financial support (grant number 00/007).
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