Robert J. North, The Trudeau Institute, 100 Algonquin Ave, Saranac Lake, NY 12983, USA. E-mail: firstname.lastname@example.org
With a view to determining whether failure of mice to resolve Mycobacterium tuberculosis (Mtb) infection is a consequence of downregulation of T helper 1 (Th1) immunity by interleukin (IL)-10, mice deleted of the gene for IL-10 were compared with wild-type (WT) mice in terms of their ability to make IL-10 mRNA, generate Th1-mediated immunity [as measured by synthesis of mRNA for interferon-γ (IFN-γ)], IL-12p40 and inducible nitric oxide synthase (iNOS), and to control lung infection. It was found that the response of WT mice to infection included a substantial and sustained increase in IL-10 mRNA synthesis in the lungs. A Th1 response in the lungs of WT and IL-10−/− mice was evidenced by a large and sustained increase in the synthesis of mRNA for IFN-γ, IL-12p40 and iNOS, with somewhat higher levels of these mRNA species being made in the lungs of IL-10−/− mice, particularly at an early stage of infection. However, IL-10−/− mice were no more capable than WT mice at combating infection.
Immunity of mice to infection with virulent Mycobacterium tuberculosis (Mtb) is mediated predominately by T helper 1 (Th1) CD4 T cells with the aid of CD8 T cells.1–3 The expression of immunity, however, is carried out by macrophages (Mφs) in which Mtb resides at sites of infection. This requires that the anti-mycobacterial function of Mφs be upregulated by Th1 cells via the secretion of interferon-γ (IFN-γ) and other cytokines. Upregulation of the Mφ anti-mycobacterial function is evidenced by their synthesis of inducible nitric oxide synthase (iNOS), which catalyses high-output generation of nitric oxide from l-arginine.4 Appropriate gene-deleted mice, incapable of making Th1 cells,3 IFN-γ5,6 or iNOS,7 are highly susceptible to Mtb infection. The Th1-mediated immunity that is generated by immunocompetent wild-type (WT) mice, however, does not provide them with the capacity to resolve infection, but enables them, instead, to gain control of bacterial growth and to hold infection at a stationary level for a protracted period of time (discussed in ref. 3). Persistent stationary lung infection, however, causes progressive lung pathology and eventually death.3 It follows therefore that in order to prevent progressive lung disease from developing it would be necessary for immunity to cause lung infection to resolve. Explaining why immunity fails to resolve infection therefore is a key problem in tuberculosis research. It is generally assumed that failure to resolve infection is a consequence of the generation of an inadequate level of Th1-mediated immunity, and it has been suggested,8,9 in accordance with the Th1/Th2 paradigm,10 that this results from downregulation of Th1 immunity by a Th2 response. However, this explanation seems unlikely, given results of a recent study11 showing that gene-deleted mice incapable of generating a Th2 response are no more capable than WT mice at dealing with Mtb infection. An alternative explanation is that anti-Mtb immunity is downregulated by the anti-inflammatory cytokine, IL-10, which is known to be produced by a variety of cell types, including T cells, dendritic cells and Mφs,12 and to be involved in downregulating immunity to infection with a variety of pathogens.13 It has been shown to be responsible for the persistence of low-level Leishmania major infection in genetically resistant mice.14
IL-10 is known to be capable of suppressing the transcriptional activation of genes necessary for the activation state of Mφs15 and of suppressing the ability of activated Mφs to inhibit the growth of the bacille Calmette–Guérin (BCG) strain of M. bovis in vitro.16 This is in keeping with the demonstration17,18 that IL-10−/− mice are more resistant to BCG infection than WT mice, which, in turn, is in keeping with the finding that BCG grows more in the organs of transgenic mice whose T cells19 or Mφs20 constitutively express IL-10. Again, IL-10 appears to be involved in the downregulation of resistance to infection with M. avium, as evidenced by an increased ability of mice treated with anti-IL-10 antibody,21,22 and IL-10−/− mice,23 to resolve infection with this organism. However, it is questionable whether findings dealing with the downregulation of immunity to BCG and M. avium can be used to explain the inadequacy of immunity to infection with virulent Mtb. Nevertheless, it has been demonstrated, by one laboratory,23 that IL-10−/− mice display an increased ability over WT mice to control growth of virulent Mtb at an early stage of infection, and that this increased resistance is associated with an increased ability of splenocytes to synthesize IFN-γ. Again, it has been demonstrated more recently24 that transgenic mice, in which IL-10 expression is under the control of the IL-2 promotor, allow a significantly higher level of growth of Mtb in their lungs than WT mice from about day 60 of infection and subsequently. However, according to another laboratory,25 IL-10−/− mice are no more capable than WT mice at controlling Mtb infection. Therefore, a role for IL-10 in downregulating Th1-mediated immunity to Mtb infection would appear to be controversial. The subject is important because the immunosuppressive action of IL-10 has been used to explain progressive tuberculosis in humans. For example, it has been shown26 that T cells capable of making IL-10, as well as IFN-γ, are more numerous in the lungs of humans with active rather than with inactive tuberculosis, and that neutralization of IL-10 with anti-IL-10 antibodies enables human peripheral blood mononuclear cells from persons with active disease to make more IFN-γ and IL-12 in response to Mtb antigens.27 Again, T cells from anergic patients with active tuberculosis, in contrast to T cells from patients with a positive skin test, have been shown28 to make IL-10, but not IFN-γ, in response to stimulation with purified proten derivative (PPD) in vitro. It seemed important, in view of this type of in vitro evidence, to look more carefully at whether the persistence of Mtb infection in mice is caused by the action of IL-10.
This study showed that the response of WT mice to aerogenic infection with Mtb includes a significant and sustained increase in synthesis of mRNA for IL-10 in the lungs. In addition, it showed that this response also includes the generation of Th1-mediated immunity, as evidenced by a significant and sustained increase in the synthesis of mRNA for IFN-γ, IL-12 and iNOS in the lungs, with higher levels of synthesis of these mRNA species in IL-10−/− mice. Finally, no difference was identified between IL-10−/− and WT mice in their ability to deal with Mtb lung infection.
Materials and methods
WT and IL-10−/− C57BL/6 male mice were obtained from The Jackson Laboratory (Bar Harbor, ME). They were used in experiments when 10 weeks of age.
Bacteria and infection
The H37Rv strain (TMC no. 102) was obtained originally from the Trudeau Mycobacterial Culture Collection (TMC) (Trudeau Institute, Saranac Lake, NY). It was grown in suspension culture in Proskauer and Beck medium containing 0·01% Tween-80, harvested during log phase [at approximately 108 colony-forming units (CFU)/ml], dispensed (in 1-ml lots) into glass vials, and frozen and stored at −70° until required. For infection, the contents of a vial were thawed, subjected to two 5-second bursts of ultrasound, and diluted appropriately in phosphate-buffered saline (PBS) containing 0·01% Tween-80. The diluted culture was then used to load the nebulizer of an aerosol-infection apparatus (Tri Instruments, Jamaica, NY). Mice were exposed to aerosol for long enough to ensure implantation in the lungs of approximately 102 CFU of H37Rv. Changes in the number of H37Rv CFU in the lungs against time of infection was determined by plating 10-fold serial dilutions of whole-lung homogenates on enriched Middlebrook 7H11 agar, and counting colonies with the aid of a dissecting microscope after 3 weeks of incubation at 37°.
Quantification of host gene expression in infected lungs by real-time reverse transcription–polymerase chain reaction (RT–PCR)
Lungs were harvested at the time-points indicated in the Results, and snap-frozen in liquid nitrogen. RNA extraction, clean-up and quantification were performed as previously described.11 To make RNA standards, amplicons for IL-10, IL-12p40, IFN-γ and iNOS were generated by PCR from WT mouse lung mRNA using the same reverse primer and T7 promoter-elongated forward primer as those used for RT–PCR. Primers and probes were designed by using Primer Express software (PE Biosystems, Foster City, CA). The sequence of each amplicon was determined by thermocycler sequencing. After agarose-gel extraction using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA), amplicons were transcribed using T7-MEGAshortscript (Ambion, Austin, TX). Template DNA was removed by digestion with DNAse I (Ambion), and the RNA was purified on RNeasy mini columns (QIAGENE) and quantified using the RiboGreen assay (Molecular Probes, Eugene, OR). It was determined that 1 µg of an average 1000-bp mRNA contained 1·8 × 1012 molecules. To obtain a standard curve, serial dilutions of each transcript were performed in triplicate to give dilutions ranging from 108 to 101 molecules. The dilutions were then subjected to real-time RT–PCR analysis, as previously described.11 Real-time PCR, to measure IL-10, IFN-γ, IL-12p40 and iNOS gene expression, was performed as described previously.11 Briefly, 1 µg of lung RNA was transcribed using a random hexamer or an oligo dT primer and a TaqMan Gold RT–PCR kit (PE Biosystems), according to the manufacturer's instructions. Real-time PCR was performed in the ABI-prism 7700 Sequence Detector (PE Biosystems) to enumerate IL-10, IFN-γ, IL-12p40 and iNOS amplicons. The copy number of RNA in each sample was calculated, as described previously,11 using the standard curves obtained as described above.
Growth of Mtb in WT versus IL-10−/− mice
The ability of IL-10−/− versus WT mice to deal with airborne Mtb infection is shown in Fig. 1. It can be seen that in both types of mice Mtb grew log-linearly in the lungs for 20 days, after which infection underwent limited resolution before being controlled at a stationary level until day 100, when the experiment was terminated. There was no significant difference between growth of the pathogen in the lungs of WT and IL-10−/− mice over the course of infection.
IL-10, IFN-γ and iNOS gene expression in the lungs of WT versus IL-10−/− mice in response to infection
Anti-Mtb immunity is mediated by Th1 cells via the secretion of IFN-γ, but is expressed by Mφs through iNOS-dependent generation of nitric oxide and reactive nitrogen intermediates.7 The generation of Th1-mediated immunity is dependent, however, on the synthesis of IL-12.29 Therefore, the kinetics and magnitude of the Th1 response to Mtb infection can be gauged by measuring changes in IL-12, IFN-γ and iNOS gene expression in the lungs during the course of infection. Moreover, to justify postulating that IL-10 is responsible for limiting the effectiveness of Th1 immunity, it was deemed necessary to show that this cytokine is, in fact, made in the lungs in response to infection. The results of an experiment that measured the synthesis of mRNA for IFN-γ, IL-12p40, iNOS and IL-10 in the lungs of WT and IL-10−/− mice against time of airborne infection, initiated with 102 CFU of H37Rv, is shown in Fig. 2. It can be seen that in the lungs of WT mice, but not IL-10−/− mice, synthesis of mRNA for IL-10 increased substantially between days 10 and 20 of infection, and remained at an elevated level until the experiment was terminated at day 100. It can also be seen that between days 10 and 20 of infection in the lungs, for both WT and IL-10−/− mice, synthesis of mRNA for IL-12p40 and IFN-γ increased substantially, as did mRNA for iNOS. Moreover, the increased levels of IFN-γ, IL-12p40 and iNOS mRNA were sustained from day 20 of infection until day 100. Kinetically, increases in the synthesis of IL-12p40, IFN-γ and iNOS mRNA were similar in IL-10−/− and WT mice. However, at day 20 of infection, IL-10−/− mice produced sevenfold more IFN-γ mRNA, threefold more IL-12p40 mRNA and fourfold more iNOS mRNA than WT mice. IL-10−/− mice also made slightly more of these mRNA species on day 50. It will be noted, in Fig. 2, that the baseline copy numbers (between days 1 and 10 of infection) of mRNA for IL-12p40, IFN-γ and iNOS were higher in the lungs of IL10−/− mice than in the lungs of WT mice.
The immunoregulatory function of IL-10 and its ability to inhibit Mφ activation and to suppress inflammatory responses, in general, are well established.12 The ability of this cytokine to decrease resistance of experimental animals to infection with intracellular pathogens has also been demonstrated.13 However, a downregulatory role for IL-10 in the immunity of mice to infection with virulent Mtb seems controversial. It is shown here that IL-10 mRNA synthesis increased substantially in the lungs of WT mice between days 10 and 20 of infection, and was sustained for the remaining 80 days of the experiment. The results also show that infection in both WT and IL-10−/− mice included a large increase in the lungs (between days 10 and 20) in the synthesis of mRNA for IL-12p40, IFN-γ and iNOS, which was sustained until the experiment was stopped. Therefore, according to IL-12p40 and IFN-γ synthesis, both types of mice generated a vigorous Th1 response. However, the Th1 response, as measured by this means, was somewhat higher in IL-10−/− mice at day 20, and possibly at day 50, of infection. Regardless, the higher level of immunity in IL-10−/− mice made no difference to the ability of these mice to deal with infection. In both types of mice, infection was not resolved but was controlled at the same stationary level of more than 6 logs from day 20 onwards. Therefore, in agreement with the results of preceding studies11 the control of Mtb lung infection at a persistent stationary level was associated with the continuous expression of Th1-mediated immunity and with the continuous expression of iNOS by Mφs. That iNOS synthesis is essential for maintaining stationary-level infection is evidenced by the finding7 that inhibiting the function of this enzyme results in the resumption of progressive Mtb growth. The need for continuous IL-12 synthesis to maintain the expression of already-established Th1-mediated immunity has been discussed in a number of publications,21,30,31 including one dealing with immunity to tuberculosis in mice.11
The finding here that IL-10−/− and WT mice display an identical capacity to control Mtb infection is in disagreement with results of others,23 which show that IL-10−/− mice were somewhat more capable than WT mice at controlling Mtb growth in the lungs at an early stage of infection. The possibility cannot be excluded, however, that these relatively small differences in the early growth of Mtb in the lungs found between laboratories are the result of differences in the ages and strains of mice used. On the other hand, the additional finding here, that a higher level of Th1-mediated immunity was generated in the lungs of IL-10−/− mice early in infection, is in agreement with the finding23 that splenocytes harvested at an early stage of infection from Mtb-infected IL-10−/− mice are more capable than splenocytes from WT mice at synthesizing IFN-γ. Even so, when interpreting the results of in vitro experiments with dissociated spleen cells, the possibility needs to be considered that the cells which make IL-10 in the spleen during infection are located at different anatomical sites from those at which immunity is being expressed by Th1 cells and Mφs. Given the well-documented immunosuppressive action of IL-10 in vitro, it seems probable that the cells that were expressing IL-10 in the lungs in response to Mtb infection, as described here, were not at the same sites at which Th1 immunity was being expressed. This line of reasoning would need to be taken into account when assessing the relevance of results of a recent publication,24 showing that Mtb grew considerably more after day 60 of infection in the lungs of transgenic mice in which IL-10 expression was under the control of the IL-2 promoter. Needless to say, in such transgenic mice, IL-10 would be produced by all cells that produce IL-2 in response to infection, a situation that is unlikely to occur in WT mice. Indeed, the results of an immunocytochemistry study of lung sections of Mtb-infected C67BL/6 WT mice in the same publication24 showed no IL-10-containing cells in lung lesions. It is unfortunate that lung sections of Mtb-infected, IL-10 transgenic mice were not examined in the same way. It should be pointed out that the hypothesis that IL-10 is responsible for failure of anti-Mtb immunity to resolve lung infection can be tested by determining whether resolution of infection occurs in mice that are incapable of making IL-10, or in mice in which all IL-10 is neutralized with an appropriate antibody. However, the same hypothesis cannot be tested by following the course of Mtb infection in transgenic mice in which IL-10 is produced in larger than normal quantities by cells from which it is normally not produced. This line of reasoning would also need to be considered in assessing the meaning of results of experiments with BCG-infected transgenic mice in which all T cells19 or all Mφs20 constitutively express IL-10. Whether results obtained with BCG can be used to explain mechanisms of immunity to infection with virulent Mtb is another question. BCG is considered avirulent on the grounds that it is progressively eliminated by host immunity and consequently does not cause disease. Virulent Mtb, in contrast, is not eliminated by immunity, and causes progressive disease.
Given the results presented here it seems reasonable to conclude that the failure of Th1-mediated immunity to resolve Mtb lung infection in mice cannot be attributed to downregulation of either the mediation or expression of Th1-mediated immunity by IL-10, even though IL-10 is made in the lungs in response to infection.
This work was supported by National Institutes of Health grants HL-64565 and AI-37844.