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Keywords:

  • Antibodies;
  • Bacterial infections;
  • Cytokines;
  • Tuberculosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL-4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL-4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti-mycobacterial α-crystallin mAb and mouse rIFN-γ, we observed a 40-fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL-4–/– BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL-4–/– mice with rIL-4 increased bacterial counts to wild-type levels and made the mice refractory to protection by IgA/IFN-γ. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti-IL-4/IgA/IFN-γ-treated and infected mice. We conclude that the action of IL-4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN-γ-induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti-IL-4, IgA antibody and IFN-γ, has potential for translation toward the passive immunoprophylaxis of tuberculosis.

Abbreviations:
Acr:

α-crystallin

BAL:

bronchoalveolar lavage

Mtb:

Mycobacterium tuberculosis

TB:

tuberculosis

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Better understanding of the regulatory role of cytokines in the pathogenesis of and protection against tuberculosis (TB) is of essential importance for the design of novel vaccines and immunotherapies. T cells in the blood of patients with active TB produce IFN-γ abundantly, despite concomitant IL-4 production 1, and can be sequestered from blood to disease sites 2. It has been proposed that the Th2 cytokine IL-4 could antagonize the protective Th1 cytokine IFN-γ response and lead to TNFα-mediated toxicity and fibrosis of lung granulomas 3. As the latter process may reduce dissemination of the infection, IL-4 apparently may have contrasting effects in TB. The pleiotropic nature of its action could explain why enhancement of the Th2 response by higher doses of BCG did not influence the protection of vaccinated mice against challenge infection 4. A human genetic study associated high concentrations of monocyte chemoattractant protein-1 (CCL2, MCP-1), which inhibits production of IL-12p40 in response to Mycobacterium tuberculosis (Mtb), with the development of active TB 5, while in situ hybridization showed downregulation of IL-4 mRNA in TB granulomas that exhibited caseous necrosis 6.

The role of IL-4 in TB has previously been ascertained in genetically IL-4-depleted mice. However, the obtained results were sharply contradictory in mouse strains of different genetic background infected with different doses of Mtb bacilli. On the BALB/c genetic background, bacterial colony forming unit (CFU) counts in the lungs of IL-4–/– mice were found to be 100-fold reduced 8 wks following intratracheal infection with 106 H37Rv strain Mtb bacilli 7. In a separate study in BALB/c mice, CFU were found to be paradoxically increased 2–7 wks following aerosol infection with 103 organisms 8. In contrast, in IL-4–/– mice on the C57BL/6 genetic background, lung CFU were not different from wild-type (WT) mice following i.n. infection with 2 × 105 BCG-Mycobacterium bovis9. Similarly IL-4–/–, IL-4Rα–/– C57BL/6 and Stat6–/– BALB/c mice infected with 2 × 102 H37Rv bacilli by aerosol had similar CFU counts as WT control mice 10, 11. However, the results in the latter paper showed, though did not note, that CFU in the liver were approximately 20-fold reduced 20 days after infection.

The regulatory influence of IL-4 has recently been demonstrated in relation to the ‘alternative activation’ of mouse macrophages 12. This process is characterized by increased expression of and phagocytosis by the mannose receptor 13, 14, which is used by tubercle bacilli for ‘safe entry’ into macrophages 15. It seems important for the pathogenesis of TB, because the action of IL-4 action is anti-inflammatory 16, 17 and reduces nitric oxide synthase expression 18 as well as IL-1 production 19. Increased expression of the mannose receptor could enhance the binding of the mannose-capped lipoarabinomannan, which blocks phagosome maturation in macrophages 20. Moreover, IL-4 could block the action of IFN-γ-induced nitric oxide (NO) against excessive inflammation 21, which is characterized by foamy macrophages and associates with the genetic susceptibility of mice to TB 22.

Our specific interest in the regulatory role of IL-4 was to ascertain if IL-4 depletion by an anti-IL-4 antibody could improve the protective effect of monoclonal IgA anti-α-crystallin (Acr) and recombinant mouse IFN-γ 23 against tuberculous infection in BALB/c mice. We considered that a possible synergy between these three treatments is plausible on assumption that they all act by modulating the response of infected macrophages from being a sanctuary toward the destruction of tubercle bacilli 24.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Combined IgA, IFN-γ and anti-IL-4 treatment of Mtb infection

The inoculation schedules for all experiments on the modulations of Mtb infection are represented in table 1. We reported previously that i.n. inoculation with mouse IgA anti-Acr (mAb TBA61) before and after Mtb infection reduces the 10-day lung infection in BALB/c mice 25. Here we tested if infection could be further reduced by i.v. inoculation with anti-IL-4 antibody. The results (Fig. 1A) show that anti-IL-4 treatment alone reduced mean log10 CFU counts from 5.36±0.38 to 3.94±0.46 (p<0.001). CFU counts following combined anti-IL-4 and IgA treatment (3.70±0.29) were not significantly lower than in mice treated with anti-IL-4 alone but were significantly lower than in mice treated with IgA alone (4.87±0.21; p<0.001). These results demonstrate that antibody-mediated depletion of IL-4 profoundly reduces the extent of early Mtb infection in the lungs of BALB/c mice.

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Figure 1. Reduction of Mtb lung infection by anti-IL-4 antibody, IgA anti-Acr mAb and IFN-γ (A, B) or genetic depletion of IL-4 (C). Columns and bars represent log10 mean ± SE CFU values (n=6) in the lungs of WT BALB/c mice 10 days (A) or 21 days (B) after i.n. infection with 1 × 106 Mtb (day 0). Anti-IL-4 (500 μg) was injected i.v. on day –1. IgA anti-Acr mAb TBA61 (50 μg/mouse) was given i.n. at –2 h and on day 2. IFN-γ (10 000 U) was given i.n. on day –2, at –2 h and on day 2. (C) Comparison of WT and IL-4–/– BALB/c mice. Mouse rIL-4 (5 μg mouse) was injected i.v. on days –2 and +22.* = differences compared with the groups inoculated with PBS alone at p < 0.001. § = differences between the indicated groups at p < 0.001.

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We next tested the lung CFU 21 days after infection of mice depleted of circulating IL-4 by i.v. administration of anti-IL-4 followed by i.n. inoculation of IgA and rIFN-γ, which extends the protection by IgA alone 23. We found that lung log10 CFU counts in mice treated with anti-IL-4 alone (4.79±0.41) and PBS controls (5.34±0.22) did not differ significantly (Fig. 1B), unlike the result at 10 days. This outcome could be due to the recovery of IL-4 levels, as anti-IL-4 was given 1 day before infection. However, the important result was that anti-IL-4/IgA/rIFN-γ treatment significantly reduced the CFU (3.65±0.38) when compared with IgA/rIFN-γ-treated mice (4.51±0.40; p=0.026). The effect of this ‘triple treatment’ was also significant when compared with anti-IL-4 (p=0.001) or with anti-IL-4/rIFN-γ (p=0.002) treatments, and the profound protection was 40-fold when compared with the PBS-treated controls (log10 CFU 3.65±0.38 vs. 5.34±0.22). This result was reproduced in a separate experiment (Fig. 1C, columns 1 and 2) in which the mean log10 CFU in the lungs of anti-IL-4/IgA/IFN-γ-treated mice (4.03±0.19) were reduced 37-fold (p<0.001) compared to PBS controls (5.5±0.48). However, this protection was found to be selective for the lungs, as the infection of spleens was not significantly reduced (results not shown). The latter result could reflect organ differences in either the extent of IL-4 depletion or in local IL-4 influence on tuberculous infection.

Modulation of Mtb infection in IL-4–/– BALB/c mice

In order to secure a complete and stable depletion, we employed mice genetically devoid of IL-4. The results 3 wks after infection show that CFU in the lungs of IL-4–/– mice (4.10±0.41) were significantly reduced when compared with concurrently tested WT BALB/c mice (5.5±048; p<0.001) (Fig. 1C). Spleen CFU (4.11±0.40) were similarly reduced (data not shown). However, IgA/IFN-γ treatment of IL-4–/– mice failed to significantly reduce the infection of either lungs or spleens any further (Fig. 1C). Reconstitution of IL-4 levels by i.v. inoculation with mouse rIL-4 restored the CFU counts to the infection level of WT mice. Furthermore, we found that the lung CFU of IL-4-reconstituted IL-4–/– BALB/c mice (unlike WT mice) were not reduced following i.n. application of IgA anti-Acr and mouse rIFN-γ. Finally, we tested the organ CFU 8 wks after infection to ascertain the duration of the IgA/IFN-γ-mediated inhibition of infection in IL-4-deficient mice. The results showed significantly lower CFU counts in treated as compared with untreated mice (Fig. 1C). Thus, IgA/rIFN-γ treatment sustained a relatively stable reduction of Mtb infection in the genetically IL-4-deficient mice.

Temporal factors affecting protection by anti-IL-4/IgA/rIFN-γ

We investigated whether repeating inoculation of the ‘triple treatment’ (see schedule in Table 1 and legend to Fig. 2) could prolong the duration of the reduced pulmonary Mtb infection in WT BALB/c mice. The efficacy of the combined anti-IL-4/IgA/IFN-γ treatment given once (as in Fig. 1) or twice (repeated in the 4th week) was compared by harvesting infected lungs at 3 and 8 wks (Fig. 2A). One regimen produced a 26.5-fold reduction of CFU at 3 wks (from 5.35±0.44 to 3.93±0.42; p=0.003), and there was no significant difference 8 wks after infection. However, repeating the anti-IL-4/IgA/IFN-γ treatment at 4 wks sustained a 7-fold reduction of CFU counts compared with the PBS control (from 5.37±0.46 to 4.56±0.70; p= 0.066).

Table 1. Inoculation schedules for the modulation of Mtb infection
Treatment, dose & routeTime schedule in relation to Mtb infection (day 0)a)Figures & groups
  1. a) d, day

TBA61-IgA50 μg i.n.–2 h, +2 d1, 2B/early
–2 h, +2 d, +5 d2A
+10 d2B/late
–3 h, +1 d3
Mouse rIFN-γ1 μg (10 000 U)i.n.–2 d, –2 h, +2 d1
–3 d, –2 h, +2 d, +5 d2A
–3 d, –2 d, +2 d2B/early
+10 d2B/late
Rat anti-IL-4500 μg i.v.–1 d1, 2A, 2B/early
+7 d2B/late
Mouse rIL-45 μg i.v.–2 d, +4 d1/IL-4KO mice
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Figure 2. Temporal factors for passive protection. (A) Extension of lung protection by repeated anti-IL-4/IgA/IFN-γ immunotherapy. Symbols represent the mean (n=6) CFU in the lungs of Mtb (1 × 106)-infected BALB/c mice that had been treated once (full squares) or twice (full triangle; repeat treatment 4 wks after infection) with IFN-γ (day –3), anti-IL-4 (day –1) and IgA/IFN-γ (–2 h, days +2 and +5). Untreated mice are indicated by open squares. (B) Lack of protection by ‘late’ immunotherapy. BALB/c mice infected i.n. with 2.5 × 105 Mtb at day 0 were given either ‘early’ (anti-IL-4 on day +7, IFN-γ on day –3, anti-IL-4 on day –1 and IFN-γ/IgA at –2 h and day +2) or ‘late’ (anti-IL-4 on day +7 and IFN-γ/IgA on day +10) treatment. Significant (p<0.001) reduction of infection in the lungs (black columns) but not spleens (open columns) was seen following early treatment. Inoculation schedules are shown in Table 1.

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The ‘standard’ anti-IL-4/IgA/IFN-γ early treatment regimen (as in Fig. 1) was next compared with late ‘therapeutic’ inoculations 7 and 10 days after infection (Fig. 2B). The harvest at 3 wks confirmed the results from Fig. 1C that the early regimen reduced CFU in the lung by 39.6-fold (from 5.36±0.38 to 3.94±0.40; p<0.001) but had no effect in spleen, while the same treatment initiated ‘late’ post-infection failed to reduce lung CFU significantly (only 3.5-fold).

Granulomatous infiltration of the lungs

Several previous studies indicated that the extent of granulomatous infiltration of the lungs following tuberculous infection of mice can be associated with either protection or pathology 3. Here we analysed histological sections of the lungs 3 wks after i.n. Mtb infection of BALB/c mice. This histological analysis was performed on the lungs of the with the CFU counts presented in Fig. 1B. The influence of anti-IL-4 administration was tested in mice co-inoculated i.n. with either IFN-γ or with IgA anti-Acr and IFN-γ. The results (Table 2) expressed as the mean of the total granulomatous area show clearly that lungs from IL-4-depleted mice had a significantly larger lung granuloma area than controls (16±4 vs. 8±2 mm2, respectively; p<0.05). This result occurred irrespective of the IgA and IFN-γ treatments, though it was most pronounced following combined treatment (21±5 vs. 2±1 mm2 in IL-4-depleted and control mice, respectively; p<0.01). However, the difference between groups given IL-4 alone or the triple treatment was not significant. Our findings suggest that IL-4 depletion-mediated enhancement of lung granuloma formation is not associated with protection, as granulomas were detected in both protected (IgA/IFN-γ-inoculated) and non-protected (PBS- or IFN-γ-inoculated) groups.

Table 2. IL-4 depletion increases granulomatous infiltration of the lungs
Intranasal inoculationsLung granuloma areaa)
No treatmentAnti-IL-4d)
  1. a) BALB/c mice (six per group) were infected i.n. with 1 × 106 CFU Mtb on day 0. CFU counts in the lungs are shown in Fig. 1b. The total granulomatous area (mean ± SE) in mm is shown. Significant differences are indicated (*p<0.05, **p<0.01).

  2. b) Inoculation i.n. with 1 μg (10 000 U) mouse rIFN-γ (–3 days, –2 h, +2 days and +5 days)

  3. c) Inoculation with 50 μg IgA anti-Acr (TBA61) and 1 μg rIFN-γ (–2 h, +2 days and +5 days)

  4. d) Injection of 500 μg anti-IL-4 antibody i.v. on day –1

None8±216±4*
rIFN-γb)6±215±3*
rIFN-γ and IgA anti-Acrc)2±121±5**

Proinflammatory mediators in bronchoalveolar lavage (BAL)

Since IL-4 depletion of BALB/c mice enhanced the granulomatous infiltration of the lungs, we decided to test the levels of proinflammatory monokines, chemokines and nitrate (a stable precursor of NO), which could be responsible for the mycobactericidal reactions of macrophages. We examined their levels in bronchoalveolar fluids harvested 8 wks after Mtb infection. The results (Table 3) show that nitrite levels in anti-IL-4/IgA/IFN-γ-treated Mtb-infected BALB/c mice were elevated 5.7-fold (p<0.01) over untreated mice. Of the cytokines tested, levels of TNF-α and IL-1β but not of IL-10 or TGF-β were significantly increased (p<0.05). Of the chemokines tested, the levels of CCL2, CCL3, CCL4 and CCL5 were significantly increased, while CCL11 and CCL22 levels (data not shown) were not significantly different from controls.

Table 3. Levels of inflammatory mediators in BAL
MediatorConcentrationsa)
Controlα-IL-4/IgA/IFN-γ-treatedb)
  1. a) Lavage fluid was harvested 8 wks after i.n. infection of BALB/c mice (six per group) with 1 × 106 Mtb.

  2. b) Treatment regimen: IFN-γ on day –3, anti-IL-4 on day –1 and IgA/IFN-γ at –2 h, +2 days, +5 days and again 4 wks post-infection.

  3. c) Nitrate (μM), cytokine (pg/mL) and chemokine (pg/mL) concentrations were measured (*p<0.05, **p<0.01).

CFU/lung565±174 × 10378±33 × 103
Nitritec)15±385±20**
Cytokinesc)TNF-αIL-1βIL-10TGF-β240±110620±110240±35120±10580±110*1300±250*230±20115±25
Chemokinesc)CCL2 (MCP-1)CCL3 (MIP-1α)CCL4 (MIP-1β)CCL5 (RANTES)940±1801200±220710±50610±1351800±460*2500±750*1100±250*1100±370*

Protection requires inoculation of IgA anti-Acr prior to Mtb infection

Further analysis addressed the hypothesis that protection by immunotherapy involves altered macrophage responses to IgA-opsonized Mtb bacilli. We pre-opsonized the Mtb organisms by incubation with IgA/anti-Acr in vitro and compared the lung CFU in IgA-inoculated and control Mtb-infected mice following i.n. infection (Fig. 3). Surprisingly, IgA opsonization of Mtb bacilli in vitro failed to diminish the 10-day pulmonary infection, while i.n. inoculation with the IgA antibody reduced the infection significantly (from 1.1 × 105 to 3.5 × 104 log10 CFU).

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Figure 3. Pre-opsonization of Mtb with IgA does not diminish lung CFU counts. BALB/c mice were inoculated i.n. with 5 × 105 Mtb bacilli (day 0) and 25 μg IgA anti-Acr mAb (–3 h and +1 day) (column: ‘Free mAb’) or with the same number of bacilli previously incubated with 1 mg/mL IgA mAb (column: ‘Ops. Mtb’) or with PBS for 60 min at 20°C. Following incubation with IgA, the Mtb bacilli were injected without further washing. The columns and bars represent log10 mean CFU ± SE values (n=6) in lungs 10 days after infection. These results were confirmed in a separate experiment.* = difference compared with the group inoculated with PBS alone at p < 0.001.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The prototype experiments demonstrating that IL-4 can play a crucial role in infection were conducted by Coffman et al., who showed that antibody neutralization of IL-4 imparts resistance to leishmaniasis in susceptible BALB/c mice 26. Here we present corroborating data demonstrating that depletion of IL-4 levels by either neutralising antibody or by gene knockout can profoundly reduce the bacillary counts in the organs of BALB/c mice infected i.n. with a relatively high dose of 106 Mtb . Anti-IL-4 antibody depletion reduced only the early (10-day) infection of the lungs, though repeated inoculation of the anti-IL-4/IgA/IFN-γ regimen could prolong protection. However, initiation of that regimen prior to infection was necessary to achieve protection. IL-4–/– mice had a much more stable (8-wk) reduction of infection in both lung and spleen. On the basis of rIL-4 reconstitution data, we concluded that the reduction of Mtb infection found in IL-4–/– mice can be attributed to the specific lack of IL-4. Abrogation of the protective effects of passive IgA antibodies in rIL-4-reconstituted mice may have resulted either from the higher levels of IL-4 than the ‘normal’ IL-4 levels in WT mice or from other alterations of the immune system due to a lack of IL-4 during development.

The Th1/Th2 paradigm, including the role of IL-4 in TB, is a matter of controversy. Rook and colleagues argue on the basis of studies in both humans and mice that IL-4 aggravates the pathology by enhancing the toxicity of TNF-α, leading to pulmonary fibrosis and chronic disease 3, 7. In contrast, North and colleagues argued categorically against the notion that IL-4 or other Th2 cytokines are responsible for the failure of Th1 cells to resolve the tuberculous infection 11. These contrasting opinions were derived from experiments in strains of mice with different genetic backgrounds and using different doses of infection. It is conceivable that a regulatory effect of IL-4 becomes involved only following high-dose infection, particularly in BALB/c mice, which have a greater tendency for IL-4 production 27, 28 and smaller granulomas 29 than C57BL/6 mice.

We tentatively interpret our results on the grounds of IL-4-mediated alternative activation of macrophages 13, not invoking the Th1/Th2 paradigm. IL-4 enhances macrophage endocytosis via the mannose receptor 3032, which binds mycobacterial lipoarabinomannan 33, thus representing a major route of Mtb entry into macrophages. Decreased expression of the mannose receptor following IL-4 depletion could explain the observed reduced infection in IL-4-depleted mice. IL-4 could also enhance the replication of tubercle bacilli by inhibition of TNF-α and NO production, which was reported previously 18, 34. We confirmed these results by demonstrating that the elevated production of nitrite (a precursor of NO) by J774 macrophages in response to IgA and IFN-γ was reversed to baseline by rIL-4 (results not shown). Other possible effects of IL-4 on macrophages may be an enhancement of iron uptake and storage 35 and stimulation of differentiation into epithelioid-like cells with poor mycobactericidal action 36.

Our results suggest that depletion of IL-4 reduces Mtb infection by mechanisms involving increased cellular infiltration of the lungs, which produced higher levels of mycobactericidal mediators. These results confirm previous reports of greater granuloma formation 7, 8 and higher NOS2 levels 11 in IL-4–/– Mtb-infected mice. As NO is mandatory for macrophage-mediated killing of tubercle bacilli, its increased levels in IL-4-depleted mice could be important in mediating the reduced Mtb infection. Our finding of a larger granulomatous area in IL-4–/– mice could be related to the previous findings that depletion of IL-4-producing NK1.1/Vα14NKT cells results in increased TNF-α production and granuloma formation following BCG infection 37.

Although acquired host resistance to tuberculous infection is mediated predominantly by CD4 and CD8 T cells, recent interest in passive antibody protection by the use of IgA mAb 38 is based on the powerful pro-inflammatory action of the IgA isotype on macrophages 39, 40. In these studies, i.n. application of an IgA anti-Acr mAb (TBA61) 41 produced short-term (9 days) protection against lung infection when applied alone 25 and longer (4 wks) protection when co-inoculated with mouse rIFN-γ 23. An important finding of the present study is that protection imparted by the i.n. inoculation of IgA and rIFN-γ can be further extended and improved by anti-IL-4 antibody treatment. However, this effect was demonstrable only in mice with partial, i.e. antibody-mediated, depletion of IL-4, but was overridden by the effect of complete depletion in IL-4–/– mice. The lack of passive protection in IL-4-reconstituted IL-4–/– mice suggests interference with IgA/rIFN-γ-mediated protection by the presumably high levels of IL-4. This result is corroborated by the finding that rIL-4 abrogates IgA/IFN-γ-induced activation of and elevated NO production by mouse macrophages in vitro (results not shown). Although our protection experiments involved passively inoculated IgA mAb, we could infer that local IgA antibody-producing B cells would require the help of Th2 cell-produced IL-4, which in turn could block the potential mycobactericidal action of the actively produced IgA antibodies on Mtb-infected macrophages.

As i.n. inoculated IgA could directly interact with macrophages, it was of interest to find out if there is also protection against in vitro pre-opsonized bacteria, as reported in studies using IgG3 42 or IgG1 43, 44 class mAb. However, we were able to demonstrate protection only when IgA anti-Acr mAb was inoculated i.n., not following the opsonization of bacilli in vitro. This result suggests that macrophage receptor occupancy by IgA antibodies prior to infection is a prerequisite for protection and argues clearly against the ‘exclusion of infection’ mechanism, which had been proposed for the action of IgG antibodies 43.

Reduced lung Mtb infection following the combined anti-IL-4/IgA/IFN-γ treatment is associated with higher levels of NO, TNF-α, IL-1β and proinflammatory chemokines in the BAL of infected mice. NO levels reflect the activity of the inducible nitric oxide synthase, which critically determines host resistance to mycobacterial infections 45. Chemokines are potent leukocyte activators and chemoattractants aiding granuloma formation in response to Mtb 46. Granuloma size appears to be regulated at least in part by IL-4 but is not coordinately regulated by bacterial load (see Table 2). The tested major beta chemokines that were elevated by the triple immunotherapy, namely CCL2, CCL3, CCL4 and CCL5 (all ligands of the chemokine receptor 5), can attract and activate macrophages and Th1 lymphocytes and were found to be elevated following BCG vaccination 47. Chemokine receptor 5 has been associated with the generation of type 1 cytokine-producing granulomas 48. Although this receptor plays a role in the migration of dendritic cells to and from lymph nodes 49, it was found to be indispensable for granuloma formation and immune protection against Mtb infection 50.

Several inhibitors of IL-4 production or action, including anti-IL-4 antibodies and soluble IL-4 receptor, are currently being developed for the treatment of asthma 51, 52. Such a strategy could also be developed and exploited for the passive immunoprophylaxis of TB. This could be particularly suitable in cases of elevated IL-4 levels, i.e. in patients with pulmonary cavities 53 and geographical areas with high IL-4 levels due to exposure to environmental mycobacteria or helminthic infections 54, which have been shown experimentally to increase susceptibility to tuberculous infection 55. Moreover, TB household contacts and health workers in Africa, who also have elevated IL-4 expression 56, may benefit from immunoprophylaxis.

In conclusion, we confirm in this paper that IL-4 depletion can reduce tuberculous infection, at least in respect to relatively high infection dose in BALB/c mice. On the basis of increased pulmonary granulomatous infiltration and proinflammatory mediators, we propose a novel interpretation for the IL-4 regulatory mechanisms based on the alternative activation of macrophages rather than the Th1/Th2 paradigm. A main novelty of this paper is the demonstration that anti-IL-4 antibodies can enhance the capacity of IgA anti-Acr antibody and IFN-γ to reduce tuberculous infection in the lungs. The proof of principle that profound protection can be imparted by this combined immunoprophylaxis raises prospects for its translation toward human treatment. Such application could be beneficial particularly in immunocompromised, i.e. HIV-infected, individuals who are at high risk of tuberculous infection. Another application could be the protection of anti-TNF antibody-treated rheumatoid arthritis and Crohn's disease patients from reactivation of TB, which cannot be fully prevented by chemoprophylaxis 57. Although the data from our mouse model indicate that treatment needs to be initiated before infection, recent results suggest that initiation of the inoculations of polyclonal anti-Mtb sera can be effective when applied in conjunction with chemotherapy 58.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice and infection with Mtb

BALB/c mice were purchased from OLAC Ltd. through Nossan (Correzzana, Italy). IL-4 gene-deficient (IL-4–/–) BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were fed and kept under specific pathogen-free conditions and used at 8 to 10 wks of age. In each experiment, six age- and sex-matched mice per group were used. Mice were infected i.n. with doses between 2 × 105 and ×106 CFU of mid-log-phase Mtb H37Rv in 0.02 mL saline given under light anaesthesia.

Modulation of the infection

All described inoculation schedules, with references to individual experiments, are summarised in Table 1. The TBA61 IgA mAb against Acr was purified from tissue culture supernatant as described previously 25. In brief, hybridoma cells were grown in CL-1000 tissue culture flasks (Integra Biosciences) in protein-free hybridoma medium (Invitrogen) supplemented with 5% fetal bovine serum. Antibody was purified from the supernatants on an Acr affinity chromatography column. Purified IgA MOPC315 myeloma protein does not differ in action from PBS 25 and therefore was not used in this study. Mice were inoculated i.n. with 50 μg TBA61 combined with inoculation with 1 µg mouse IFN-γ (10 000 U/μg, Serotec Oxford, UK) in 25 µL saline. For IL-4 depletion, BALB/c mice were injected i.v. with 500 μg IL-4-neutralising rat mAb to mouse IL-4 (clone 11B11, Becton Dickinson Biosciences).

IL-4 reconstitution of IL-4–/– BALB/c mice

Each mouse was injected i.v. with 5 μg mouse rIL-4 complexed with an anti-IL-4 antibody, which stabilises and prolongs the action of IL-4, as described previously 7. Briefly, 5 μg rIL-4 (Santa Cruz Biotechnology, CA) mixed with 5 μg polyclonal rat anti-mouse IL-4 antibody (R&D Systems, Minneapolis, MN), each in 20 μL saline solution, were incubated for 5 min at room temperature and then diluted with saline to a total volume of 100 μL.

CFU and histology

Lungs and spleens were harvested from Mtb-infected mice at different time points, and 0.15 mL of serial 10-fold dilutions of whole spleen or lung lobe homogenates were plated on Middlebrook 7H11 agar plates. For histological analysis, organs were fixed in 10% normal buffered formalin and embedded in paraffin, and 5-μm sections were stained with haematoxylin and eosin. The area of granulomatous infiltration (mm2) was measured using a Zeiss image analysis system. Measurements were done blindly by two independent pathologists.

Assays for nitrite, cytokines and chemokines

BAL was obtained by flushing 2 mL PBS into the lungs of killed mice. The concentration of nitrite, a stable metabolite of NO, was determined using Griess reagent (Sigma) by measuring absorbance at 550 nm in a micro-ELISA reader. The concentrations of cytokines (TNF-α, IL-1β, IL-10, IFN-γ, TGF-β) and chemokines [CCL2 (monocyte chemotactic protein-1, MCP-1), CCL3 (macrophage inflammatory protein-1α, MIP-1α), CCL4 (MIP-1β), CCL5 (regulated on activation, normal, T-cell expressed and secreted, RANTES), CCL11 (eosinophil-attracting chemokine, Eotaxin), CCL22 (MDC)] were measured by ELISA kits (R&D Systems) according to the manufacturer's recommendations.

Statistical analysis

The significance of differences between groups was determined using one-way analysis of variance (ANOVA) of log10 CFU counts, with Scheffe tests for post-ANOVA individual comparisons. Student's t-test was used for the analysis of granuloma, NO, cytokine and chemokine values in the lungs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Funding was obtained from the University of Palermo, the Italian Ministry for University and Research and the Dunhill Medical Trust, London. We also thank Pascal Drake and Ron Wilson for help with statistical evaluation.

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