Thomas Merlin

Cutting Edge: A Murine, IL-12-Independent Pathway of IFN-γ Induction by Gram-Negative Bacteria Based on STAT4 Activation by Type I IFN and IL-18 Signaling

IFN-αβ is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-αβ-dependent pathway of IFN-γ induction in mice. This pathway is based on a synergism of IFN-αβ and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-αβ alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-γ by Gram-negative bacteria and is therefore expected to play a role whenever IFN-α or IFN-β and IL-18 are produced concomitantly during bacterial, viral, or other infections.

Bacterial Induction of Beta Interferon in Mice Is a Function of the Lipopolysaccharide Component

ABSTRACT We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lpsd]) C57BL/10ScCr mice to produce beta interferon (IFN-β) when stimulated with bacteria. For this purpose, the IFN-β and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lpsn]; C57BL/10ScSn and BALB/c) and Lpsd (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-β in the Lpsn mice but not in theLpsd mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-β in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1α [IL-1α], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-β and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-β in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-β is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

A Point Mutation in the IL-12Rβ2 Gene Underlies the IL-12 Unresponsiveness of Lps-Defective C57BL/10ScCr Mice

Lps-defective C57BL/10ScCr (Cr) mice are homozygous for a deletion encompassing Toll-like receptor 4 that makes them refractory to the biological activity of LPS. In addition, these mice exhibit an inherited IL-12 unresponsiveness resulting in impaired IFN-γ responses to different microorganisms. By positional cloning methods, we show here that this second defect of Cr mice is due to a mutation in a single gene located on mouse chromosome 6, in close proximity to the Igκ locus. The gene is IL-12Rβ2. Cr mice carry a point mutation creating a stop codon that is predicted to cause premature termination of the translated IL-12Rβ2 after a lysine residue at position 777. The truncated β2 chain can still form a heterodimeric IL-12R that allows phosphorylation of Janus kinase 2, but, unlike the wild-type IL-12R, can no longer mediate phosphorylation of STAT4. Because the phosphorylation of STAT4 is a prerequisite for the IL-12-mediated induction of IFN-γ, its absence in Cr mice is responsible for their defective IFN-γ response to microorganisms.

Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse.

Lpsd mouse strains are characterized by the presence of a defective Lps/tlr4 gene that make them refractory to the biological activity of LPS. One of the mouse strains commonly used to study LPS defects is the C57BL/10ScCr (Cr) strain. However, unlike other Lpsd strains, the Cr strain also has a heavily impaired IFN-γ response to micro-organisms. As a consequence, unlike other Lpsd mouse strains, they do not acquire a partial LPS susceptibility when treated with sensitizing bacteria. Because IL-12 is important for the microbial induction of IFN-γ, we investigated whether the production or function of IL-12 might be defective in Cr mice. IL-12 mRNA (p35 and p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was inducible in mice injected with live or killed Salmonella typhimurium, and IL-12 (p70) was inducible in macrophages by bacteria. Thus, Cr mice exhibit normal IL-12 responses. In functional tests, splenocytes of untreated or of S. typhimurium-infected mice failed to produce IFN-γ when stimulated with murine rIL-12 or with a combination of IL-12 and murine rIL-18 or Con A. Furthermore, Cr mice were identical with IL-12p35/p40 and IL-12 receptor β1 knockout mice in their impaired in vivo and in vitro IFN-γ responses to bacteria. Thus, Cr mice carry a second genetic defect unrelated to the Lps/tlr4 mutation that underlies the IL-12 unresponsiveness and contributes to the LPS resistance and impaired innate immune response in this strain.

TNF-α hyper-responses to Gram-negative and Gram-positive bacteria in Propionibacterium acnes primed or Salmonella typhimurium infected mice

IFN-γ-dependent hypersensitivity to LPS is inducible in mice by infection or pre-treatment with killed bacteria. Hypersensitive mice exhibit enhanced inflammatory responses to LPS, including the overproduction of TNF-α. Using Lpsn BALB/c and Lpsd BALB/c/l mice, primed with Propionibacterium acnes or infected with Salmonella typhimurium, we show that concurrently to hypersensitivity to LPS, a hypersensitivity to other constituents of killed Gram-negative or Gram-positive bacteria and to staphylococcal enterotoxin B (SEB) develops. The TNF-α hyper-responses in sensitized mice induced by different Gram-positive bacteria, are generally weaker than those by Gram-negative bacteria and vary significantly, due to the absence of a common, LPS-equivalent component. Using IFN-γR—/— and the respective wild-type mice, we demonstrate that although sensitization to LPS and killed Listeria monocytogenes is exclusively IFN-γ-dependent, an IFN-γ-independent, moderate sensitization to certain TNF-α-inducing constituents in bacteria may develop in parallel.

Role of interferons in LPS hypersensitivity.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-γ-dependent and moderate IFN-γ-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-γ by bacteria. We showed that Gram-negative bacteria induce IFN-γ in mice also by an IFN-β-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-β. Further, IFN-β can be replaced by IFN-α. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-β. Therefore, the IFN-β pathway of IFN-γ induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-α/β-dependent pathway is expected to play a role whenever IFN-α or IFN-β, and IL-18 are produced concomitantly during infection.

Expression and role of CD14 in mice sensitized to lipopolysaccharide by Propionibacterium acnes

Propionibacterium acnes‐primed mice develop an IFN‐γ‐dependent hypersensitivity towards LPS. Since CD14 plays a key role in LPS‐induced cell activation the regulation and function of CD14 in this sensitization process were studied in IFN‐γR–/– and the respective wild‐type (wt) mice. In unprimed mice, CD14 (mRNA and protein) was either absent (liver) or only weakly expressed in organs (spleen, lung) and in plasma. Priming with P. acnes led to a moderate, mainly IFN‐γ‐dependent up‐regulation of CD14. LPS challenge of unprimed mice induced an IFN‐γ‐independent increase in CD14 mRNA and CD14 protein. LPS challenge of P. acnes‐primed mice induced a strong CD14 overexpression. This response was completely absent in IFN‐γR–/– mice and is therefore strictly IFN‐γ‐dependent. The requirement for CD14 in LPS hyper‐responsiveness was assessed by comparing CD14–/– and the respective wt mice with respect to their ability to produce TNF and IFN‐γ, two recognized indices of LPS activity. LPS challenge without priming led to a weaker cytokine reaction in CD14–/– than in wt mice. However, priming with P. acnes enhanced the cytokine response to LPS in both wt and CD14–/– mice, although in the latter absolute levels of cytokines were lower. Therefore, hyperreactivity to LPS is characterized by an up‐regulation of CD14, but the sensitization by P. acnes is not CD14 dependent.

Bacteria-induced hypersensitivity to endotoxin

Endotoxin (LPS) hypersensitivity may be induced in mice by live or killed Gram-negative and Gram-positive bacteria. It is characterized by an overproduction of pro-inflammatory cytokines in response to LPS and by an enhanced susceptibility of the mice to the lethal activity of LPS and TNFα. The induction of LPS hypersensitivity by bacteria is mediated by IFN-γ. Consequently, its development can be inhibited by anti-IFN-γ antibodies and is absent in mice with an impaired IFNγ receptor. In sensitized mice, the enhanced activity of LPS is strictly LBP-dependent. Bacteria-treated, LBP-deficient mice exhibit only a marginally enhanced LPS responsiveness. In such mice, the administration of exogenous LBP at the time of LPS challenge restores the LPS hyper-response. Mice hypersensitive to purified LPS are also hypersensitive to whole Gram-negative bacteria. Such mice, however, are only moderately sensitized to Gram-positive bacteria or to the bacterial superantigen SEB. This sensitization, in contrast to the LPS and Gram-negative bacteria hypersensitivity, is IFN-γ independent. The role of LPS hypersensitivity for the outcome of Gram-negative infection is a dual one. At the early stages of infection, hypersensitivity enables the host to sense minute quantities of bacterial antigens and react against the infection. A failure of this early protective mechanism to eliminate the invading bacteria may have disastrous consequences, since the threshold of development of endotoxin shock is considerably lower in the hypersensitive host.

A strict requirement for LBP in the TNFα hyper-response of Propionibacterium acnes-sensitized mice to LPS

One important feature of the Propionibacterium acnes-induced hypersensitivity to LPS is the enhanced production of TNFα induced in the sensitized mice upon LPS challenge [Katschinski T., Galanos G., Coumbos A., Freudenberg M.A. Gamma interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect Immun 1992; 60: 1994-2001]. We investigated the role of LPS binding protein (LBP) in the TNFα response of normal and P. acnes-sensitized mice to LPS in LBP+/+ and LBP-/- mice. Treatment of LBP+/+ (BalbC and 129 Sv) mice with P. acnes enhanced their TNFα response to LPS by 75-200-fold compared to the non-treated controls. Unsensitized LBP-/- (129 Sv x BalbC) mice were also stimulated by LPS to produce low amounts of TNFα. These were enhanced by prior P. acnes treatment, however, only by a factor of 4. The characteristic TNFα hyper-response was absent suggesting that the enhanced activity of LPS in sensitized mice is expressed only in the presence of LBP. Evidence for this was obtained by showing that administration of exogenous LBP restored fully the inducibility of the TNFα hyper-response in P. acnes-sensitized LBP-/- mice. Their response to LPS was 1000-fold higher then that of sensitized controls without LBP. A similar LBP treatment of unsensitized LBP-/- mice increased the TNFα response by a factor of only 5. In an analogous experiment, also IFN-γ-sensitized LBP-/- mice exhibited a TNFα overproduction in response to LPS only in the presence of exogenous LBP.