eNews July 12, 2011

Tlr9 and Ifng are required to induce a 'cytokine storm' syndrome in mice

A strong immune system is a good thing, but too much of a good thing may not be so good after all. Sometimes, the immune system may overreact when it encounters new or particularly pathogenic agents - such as the viruses that caused the 1918 flu pandemic, the 2003 SARS epidemic in Hong Kong or, more recently, the 2009 swine flu pandemic. When the immune system encounters such agents, its normal cytokine activation feedback loop may be severely disrupted, producing a "cytokine storm" that leads to overwhelming and sometimes fatal inflammation and multi-organ failure. While some cytokine storms have known causes, others are triggered for no apparent reason.

In 2011, a research team led by Edward Behrens. M.D., at The Children's Hospital of Philadelphia and Gary Koretzky, M.D., Ph.D., of the Perelman School of Medicine at the University of Pennsylvania, reported that one of the underlying triggers may be the repeated stimulation of toll-like receptor 9 (TLR9) (Behrens et al. 2011). The research may lead to the development of new therapies for inflammatory diseases characterized by chronic innate immune activation in the absence of genetic cytotoxic T-cell defects.

Cytokine storms

Cytokine storms are caused by a number of underlying triggers, but each trigger has similar outcomes, including elevated cytokine levels, hemophagocytosis (phagocytosis of RBCs, WBCs, and platelets by macrophages), multi-organ failure and, sometimes, death. Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are two related diseases characterized by cytokine storms. In both, the elevated cytokine is primarily interferon gamma (IFNG). Generally, in humans and animal models, the elevated IFNG levels result from the inability of defective CD8+ T cells to clear a pathogen. However, HLH and MAS cytokine storms occasionally occur in the absence of defective CD8+ T cells, a pathogen or other triggers. Behrens, Koretzky and their colleagues sought to identify these triggers.

TLR9 stimulation induces MAS-like syndrome in mice

The Behrens/Koretzky team was aware of studies implicating TLRs in cytokine storm diseases. In particular, one study reported that repeated stimulation of TLR9 with CpG DNA produces systemic toxicity. To determine if repeated TLR9 stimulation could produce a cytokine storm in the absence of antigen, the researchers repeatedly stimulated C57BL/6J mice (B6J, 000664) with CpG. They found that these mice develop an MAS-like syndrome that includes leukopenia, splenomegaly, elevated serum ferritin, liver disease, disrupted bone marrow architecture and elevated IFNG, IL12p70, IL6 and IL10 levels. Attempts to produce the same effects in either Tlr9-deficient or Ifng-deficient – B6.129S7-Ifngr1tm1Agt/J (003288) – mice failed, indicating that the CpG-induced MAS-like disease in their model is mediated through TLR9 stimulation and is at least partly caused by elevated IFNG levels.

Lymphocytes and other cells are the IFNG producers

B6.129S7-Ifngr1tm1Agt/J mice are Ifng-deficient and do not develop CpG-induced MAS.

To identify the immune cells responsible for producing the excess IFNG, the researchers examined the CpG-induced MAS response in several immunodeficient mouse models. They observed the following:

  • CpG treatment induces MAS but only minimally activates CD8+ T cells in B6J mice.
  • B6.129P2-B2mtm1Unc/J (002087) mice, which are deficient in both CD8+ T and NKT cells, develop a CpG-induced MAS similar to that of control B6J mice.
  • CpG-treated C57BL/6-Rag2tm1Cgn/J mice (008309), which lack B and T cells, develop a similar CpG-induced MAS.
  • CpG-treated NK-depleted B6J mice also develop a similar MAS.
  • CpG-treated mice deficient for both Rag2 and Il2rg (and which therefore lack B, T, NKT and NK cells) develop severe hepatitis, but an attenuated CpG-induced MAS.

To the researchers, these results suggested that though both NK cells and T and/or B cells play a role in IFNG production and maximum CpG-induced MAS, non-lymphocyte cells also play a key role.

Liver and spleen dendritic cells also produce IFNG

To determine which non-lymphocyte cells produce IFNG in CpG-induced MAS, the researchers used a Yeti IFNG reporter mouse. IFNG-producing cells in this mouse can be identified because they express yellow fluorescent protein. The researchers found that one CpG treatment in these mice induces a small increase in IFNG production in two subpopulations of liver dendritic cells (DCs): conventional DCs (cDCs) and plasmacytoid DCs (pDCs). However, repeated CpG treatments induce a dramatic increase in IFNG production in both DC subpopulations – not in the liver but in the spleen. To determine the relative IFNG contributions of the two DC subpopulations, the researchers analyzed IFNG production and MAS severity in cDC-depleted mice. To produce mice with long-term cDC depletion, they transferred bone marrow from diphtheria toxin-treated B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J mice (004509) (the toxin deletes cDCs – but not pDCs – in these mice) to B6J mice. They found that though the cDC-depleted B6J mice produce no IFNG after one CpG injection, they do produce it – and develop cytopenia, splenomegaly and hepatitis – after repeated injections. This indicated that though cDCs are responsible for the initial INFG production, they are not needed to produce the later-phase INFG and to precipitate MAS disease.

IL10 mitigates CpG-induced MAS

In the course of their study, the researchers had observed that the CpG-induced MAS in their model is relatively mild and accompanied by considerably elevated levels of IL10, a recognized anti-inflammatory cytokine. So, they examined IL10 production in CpG-treated Ifng-deficient mice and found that these mice produce much less IL10 than controls. To determine if lymphocytes produce the IL10, the researchers examined IL10 levels in CpG-treated Rag2-deficient mice. They found that, in addition to having very low IL10 levels, these mice develop severe liver damage, suggesting that the absence of lymphocytes (and the IL10 they produce) exacerbates CpG-induced MAS. The researchers further investigated the role of IL10 by treating B6J mice with both CpG and an IL10 blocker. They found that these mice develop a more severe MAS than B6J mice treated with only CpG, indicating that IL10 considerably ameliorates CpG-induced MAS.

In summary, cytokine storms are characterized by elevated cytokine levels, hemophagocytosis and multi-organ failure, and they are sometimes fatal. They can be caused by defective antigen-specific responses of either B or T cells, by non-antigen-specific inflammation from pattern recognition receptors (including TLRs) or by unknown triggers. Behrens, Koretzky and their colleagues demonstrated that the CpG-induced MAS-like cytokine storm in their model is due to excess IFNG production caused by repeated TLR9 stimulation, does not require exogenous antigen and is ameliorated by IL10. Their findings may lead to the development of new therapeutic targets for cytokine storms that occur in the absence of genetic defects in cytotoxic T cells.