1 Immunovirology Laboratory, Institute of Immunology and Biology Department, National University of Ireland, Maynooth, Co. Kildare, Ireland
2 National Institute for Biological Standards and Controls, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK
3 Mucosal Immunology Laboratory, Institute of Immunology, National University of Ireland, Maynooth, Co. Kildare, Ireland
4 Immune Regulation Research Group, Biochemistry Department, Trinity College Dublin, Ireland
Correspondence
Patricia A. Johnson
p.johnson{at}may.ie
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ABSTRACT |
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MAIN TEXT |
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Studies have demonstrated a role for autocrine production of IL10 in the suppression of IL12 responses in vivo and in vitro (Aste-Amezaga et al., 1998; McGuirk et al., 2002
). To further elucidate the mechanism underpinning IL12 downregulation by influenza virus HA, we explored the possibility that IL10 may mediate the effect. We demonstrate that HA does not induce IL10 alone, nor does it synergize with LPS to produce elevated levels of this cytokine. Furthermore, pre-incubation with an anti-IL10 antibody did not revert the suppressive effect of HA on LPS-induced IL12 production by BMDCs (Fig. 2b
). It has also been demonstrated that, during viral infections, early type 1 alpha and beta interferons (IFN-
/
) downregulate IL12 p70 production (Cousens et al., 1997
). However, the lack of effect on other cytokines, particularly IL12 p40, which is negatively regulated by IL10 (Aste-Amezaga et al., 1998
; McGuirk et al., 2002
), and the inability to block the effect with anti-IL10 antibodies suggest that a cytokine intermediate is not involved in the downregulation of LPS-induced IL12 production by HA. Similar to our finding with anti-IL10, we could not restore IL12 p70 production by pre-incubation with an anti-IFN-
/
antibody (data not shown).
Bioactive IL12 differs from other pro-inflammatory cytokines in that it is a heterodimer, comprising 35 kDa (p35) and 40 kDa (p40) polypeptides encoded by separate genes (O'Garra, 1998; Trinchieri, 1998
). Although both subunits must be co-expressed within the same cells to generate the bioactive form, evidence has suggested that expression of each subunit is regulated independently (Murphy et al., 1995
). It has been suggested that p40 expression controls the level of bioactive IL12, as this subunit is highly regulated, whereas p35 is expressed ubiquitously (Snijders et al., 1996
). However, in our study, the levels of p40 protein remained elevated, despite the downregulation of the heterodimer p70. These findings lend weight to other reports that attest to the regulation of p35 and suggest that p35 is the limiting factor in bioactive IL12 production, as the level of p40 exceeds that of p35 (Gillessen et al., 1995
; Heinzel et al., 1997
). In addition, the finding that p40 is frequently produced in excess as a homodimer [(p40)2] that can bind to the IL12 receptor and antagonize IL12-mediated function suggests that the biological activity of IL12 may be determined in part by the ratio of p40 to p70 (Gillessen et al., 1995
; Heinzel et al., 1997
). This, in turn, would indicate that p35 levels are likely to be an important limiting factor in determining bioactive IL12 production. In the light of earlier reports regarding p40 expression, the molecular mechanisms of its regulation have been well-characterized (Gillessen et al., 1995
; Heinzel et al., 1997
; Murphy et al., 1995
). In contrast, the mechanisms underlying p35 expression in health and disease remain largely ill-defined.
To further examine the effects of HA on the expression of both subunits of the heterodimer, the level of p40 and p35 mRNA expression was examined by RT-PCR. For RT-PCR studies, total RNA was harvested from cells at 2, 4, 6 and 24 h and the level of expression of IL12 p40 and IL12 p35 mRNA was determined by RT-PCR, using published primer sequences. The presence of HA in LPS-stimulated cultures of BMDCs did not alter p40 expression, but completely abrogated LPS-induced p35 mRNA expression up to 4 h post-treatment (Fig. 2c). p35 expression was restored at 24 h in these samples, although protein levels still demonstrated significant reduction in IL12 p70 at 24 h. This was possibly due to the delay in generating protein from mRNA and could be considered to be consistent with inhibition of mRNA transcription for at least one-third of the total treatment period. This selective inhibition of p35 gene expression is a novel and unexplored pathway to downregulate IL12 responses. These findings shed light on the control of bioactive IL12 p70 expression in DCs. The speed of inhibition of p35 expression by HA argues against the possibility of regulation by autocrine expression of cytokines, but suggests that HA interferes directly with the pathway of p35 gene activation. Furthermore, it rules out possible interference at the level of receptor binding, as intracellular signalling pathways for p40 and expression of other cytokines are unimpaired. Reports have suggested that p35 protein expression is regulated at both the transcriptional and translational levels (Grumont et al., 2001
; Murphy et al., 1995
). Although translational control cannot be ruled out clearly in this system, the complete abrogation (at 2 and 4 h) of p35 mRNA by HA is due to transcriptional control. A recent report has shown that the Rel/NF-
B signalling pathway is required for the induction of IL12 in activated CD8+ splenic DCs (Grumont et al., 2001
). Impaired expression of IL12 p70, but not p40 or (p40)2, in c-rel/ CD8+ DCs is due to an inability to upregulate p35 transcription, which is c-rel-dependent (Grumont et al., 2001
). The similarity in cytokine-expression profiles between LPS-stimulated c-rel/ DCs and LPS-stimulated, HA-treated BMDCs leads us to speculate that HA may target c-Rel-dependent transcription. Furthermore, in comparison with c-Rel/ DC studies, HA does not interfere with or disrupt normal DC maturation, whilst IL12 expression (p70, p35) is impaired (Grumont et al., 2001
). To establish whether HA exerts its suppressive effect in vivo as well as in vitro, groups of four BALB/c mice were injected intravenously with HA (10 µg per mouse) and LPS (1 µg per mouse) alone or in combination and serum concentrations of IL12 and IFN-
were assessed by ELISA at 6 h post-injection. Mice were rested for 1 h between each dose. Consistent with DC studies, HA significantly downregulated LPS-induced IL12 p70 production in mice, which was accompanied by a reduction in IFN-
levels (Fig. 3
).
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To our knowledge, no other study has demonstrated immunosuppressive properties for HA, the major surface antigen of influenza virus. Whilst studies have defined a clear role for IL12 in the development of Th1 responses to bacterial infections, such a monopoly for IL12 in the induction of the Th1 response to viral infection is less definitive. Several recent studies have demonstrated that mice lacking IL12 can develop polarized Th1 cells during viral infection (Oxenius et al., 1999; Schijns et al., 1998
). In addition, it has been reported that influenza virus infection fails to induce p35 or p40 mRNA expression in human macrophages (Pirhonen et al., 2002
). We also observe identical Th1 responses from IL12-knockout mice compared with wild-type controls, following challenge with live influenza virus (C. M. Noone & P. A. Johnson, unpublished observations). This begs the question as to the likely advantage that the inhibitory effect of HA may have for virus survival. Increasingly, studies have revealed the enormous redundancy that is built into the immune system and that, whilst a virus may subvert one pathway, which may historically have prolonged its survival, the host, likewise, may have evolved other means to eliminate the infecting virus. An alternative and possibly more likely explanation is that the p35 pathway is also involved in some as-yet-unidentified pathway of virus clearance and that, whilst p35 expression may be redundant in antiviral responses, the p35 pathway may not be. Studies carried out in IL12-knockout mice have traditionally targeted the p40 pathway, so the influence of the pathway that leads to p35 expression on antiviral immunity remains to be explored. Thus, inhibition of this pathway may confer some selective advantage on the virus, independent of type 2 IFN production. Influenza is a successful pathogen: even beyond its ability to generate new antigenic types, it possesses other immune-subversive tactics, such as the inhibition of the type 1 IFN pathway in myeloid-derived DCs by the non-structural protein of the virus (Marcus et al., 2005
). It is plausible that other viral proteins may also be involved in immune subversion/modulation.
These results suggest that, for influenza virus infection, bioactive IL12 p70 is not an absolute requirement for a polarized Th1 response during infection; however, downregulation of this cytokine may render the host more susceptible to bacterial colonization. We therefore propose that the mechanisms underlying bacterial superinfection in viral respiratory disease are not simply a consequence of increased bacterial adherence, but involve more complicated physiological mechanisms relating to the immunomodulating properties of key viral antigens. We hypothesize that virus components alter the course of critical immune responses to bacterial invasion, rendering the host more susceptible to infection. Our finding that HA from influenza virus modulates IL12 production by BMDCs represents a novel and unexplored pathway by which influenza virus may render the host more susceptible to bacterial infections.
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Received 13 January 2005;
accepted 12 April 2005.
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