Repeated TLR9 stimulation results in macrophage activation syndrome–like disease in mice

Second-phase IFN-γ after day 3 is crucial to the development of pathology.

“This observation demonstrates that although the amount of IFN-γ seen at the later time points is modest, it is crucial to the development of pathology.”

CpG-treated mice develop microthrombi in liver small vessels with adjacent lobular necrosis.

“Three out of six livers from CpG-treated mice developed thrombi in their small vessels, with lobular necrosis adjacent to these vessels (Figure 1F).”

Repeated CpG treatment elevates serum IFN-γ at day 10.

“Mice treated with multiple doses of CpG also demonstrate elevated serum IFN-γ at sacrifice on day 10 after initial CpG treatment (Figure 2A).”

IFN-γ is required for maximal disease expression in repeated CpG-treated mice.

“IFN-γ is required for maximal disease expression in repeated CpG-treated mice.”

• Also stated in: § Repeated TLR stimulation leads to the clinical appearance of the cytokine storm syndrome. · boosted

  “Mice that received IFN-γ neutralization throughout the entire experiment phenocopied the Ifng -/-mice, confirming that IFN-γ production is critical for development of disease.”

• Also stated in: § Repeated TLR stimulation leads to the clinical appearance of the cytokine storm syndrome. · plain

  “Compared with wild-type counterparts, Ifng -/-mice did not develop anemia or thrombocytopenia (Figure 3, A and B), had markedly reduced splenomegaly (Figure 3C), did not develop hepatic inflammation (Figure 3D), and manifested preservation of splenic architecture (data not shown).”

TNF-α is not necessary for the development of the MAS-like syndrome.

“TNF-α-neutralized mice did not show any decrease in any disease parameters compared with mice receiving isotype control antibody (data not shown), suggesting that TNF-α is not necessary for the development of the MAS-like syndrome.”

CpG-treated mice develop elevated serum ferritin after 10 days of treatment.

“As in the MAS syndrome, CpG-treated mice developed elevated serum ferritin (Figure 1E) after 10 days of treatment.”

Repeated TLR9 stimulation with CpG in mice produces an HLH/MAS-like syndrome on a normal genetic background without exogenous antigen.

“Herein, we show that repeated stimulation of TLR9 produced an HLH/ MAS-like syndrome on a normal genetic background, without exogenous antigen.”

• Also stated in: § Repeated TLR stimulation leads to the clinical appearance of the cytokine storm syndrome. · plain

  “In summary, repeated TLR9 activation, even in the absence of foreign antigen, can recapitulate multiple serologic and histologic features of the MAS cytokine storm syndrome.”

• Also stated in: § Introduction · plain

  “In agreement with the report by Heikenwalder et al. of systemic toxicity (11), we found that mice treated with repeated TLR9 agonism developed cytopenia, splenomegaly, hep-atitis, microthrombosis, and hyperferritinemia, consistent with MAS-like syndromes (13).”

Livers from CpG-treated mice show a lymphohistiocytic infiltrate consisting predominately of macrophages and to a lesser degree a mixture of T and B cells.

“Livers from CpG-treated mice showed a lymphohistiocytic infiltrate consisting predominately of macrophages and to a lesser degree a mixture of T and B cells (Figure 2E).”

Repeated CpG stimulation does not increase the numbers of hemophagocytes in the bone marrow, spleen, or liver.

“After repeated CpG stimulation, we did not find an increase in the numbers of hemophagocytes in the bone marrow, spleen, or liver.”

Multiple doses of CpG are required to induce the complete disease of pancytopenia, splenomegaly, hyperferritinemia, microthrombosis, and hepatitis.

“Thus, it appears that multiple doses of CpG are required to induce the complete disease of pancytopenia, splenomegaly, hyperferritinemia, microthrombosis, and hepatitis.”

CpG-treated mice develop marked splenomegaly.

“CpG-treated mice also developed a marked splenomegaly (Figure 1C).”

CpG-induced disease is mediated through TLR9 stimulation.

“we attempted to induce disease in TLR9-deficient (Tlr9 -/-) mice but found that CpG-treated Tlr9 -/-mice were indistinguishable from mice injected with PBS (Supplemental Figure 2 and data not shown), thus demonstrating that this CpG-induced disease is mediated through TLR9 stimulation.”

Repeated CpG administration to mice results in peripheral pancytopenia.

“Repeated administration of the TLR9 agonist CpG to mice resulted in a peripheral pancytopenia (Figure 1A, rbc indices shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI43157DS1).”

Repeated CpG treatment produces two phases of IFN-γ production: an initial high burst followed by a plateau of lower amounts.

“Thus, there appear to be 2 phases of IFN-γ production, an initial high burst followed by a plateau of lower amounts of IFN-γ.”

CpG-treated mice develop lobular and portal inflammation in all livers examined.

“HLH/MAS is often associated with hepatitis, and CpG-treated mice developed both lobular and portal inflammation in all of the livers examined (Figure 2, B-D).”

CpG-initiated MAS is accompanied by only minimal CD8+ T cell activation.

“CpG-initiated MAS is accompanied by only minimal CD8 + T cell activation.”

Repeated CpG treatment does not produce significant serum TNF-α.

“In contrast, we did not find significant amounts of serum TNF-α (data not shown).”

NK cell numbers decrease more than 20-fold in the spleens of mice after repeated CpG treatment.

“NK cell numbers showed a greater than 20-fold decrease in the spleens of mice that received repeated CpG treatment (Figure 6A), suggesting either that they were activated and exiting the spleen or that they were being consumed in the process of disease progression.”

In Yeti IFN-γ reporter mice, subpopulations of pDCs and CD8α+ cDCs in the liver show IFN-γ transcription at baseline in PBS-treated mice.

“In all PBStreated mice tested, we noted subpopulations of plasmacytoid DCs (pDCs) and CD8α + cDCs in the liver that showed IFN-γ transcription as marked by YFP positivity, suggesting that these cells are transcribing IFN-γ message at baseline (Supplemental Figure 6).”

Rag2-/-Il2rg-/- CpG-treated mice show an approximate 50% reduction in IFN-γ levels on day 10 of disease.

“Rag2 -/-Il2rg -/-CpG-treated mice demonstrated an approximate 50% reduction in the levels of IFN-γ measured on day 10 of disease (Figure 7C).”

The severity of MAS in this model is less than that seen in the perforin-deficient model.

“The severity of MAS in this model is less than that seen in the perforin-deficient model (8).”

• cites [8] Jordan, 2004, Blood. sourceNot checked yet

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NK cells are dispensable for CpG-induced disease.

“NK cell-depleted mice developed cytopenia, splenomegaly, hyperferritinemia, hepatitis, and elevated IFN-γ lev- els similar to isotype antibody-treated controls (Supplemental Figure 5, B and C, and data not shown), suggesting that NK cells were dispensable.”

B2m-/- mice consistently produce more IFN-γ than wild-type mice after repeated CpG.

“In fact, we found that B2m -/-mice consistently produced more IFN-γ than wild-type mice, suggesting that the absence of CD8 + and NKT cells enhances the repeated TLR9-stimulated phenotype (data not shown).”

CpG-treated mice deficient in IFN-γ make less IL-10 than wild-type mice.

“This IL-10 is produced partly in response to the TLR9-induced IFN-γ, since CpG-treated mice deficient in IFN-γ make less IL-10 than wild-type mice.”

After a single CpG injection, there is a small but consistent increase in YFP intensity in liver CD8α+ cDCs and pDCs, consistent with increased IFN-γ transcription.

“In mice that received 1 dose of CpG (i.e., the early phase of IFN-γ production), there was a small but consistent increase in YFP intensity in liver CD8α + cDCs and pDCs, consistent with an increase in IFN-γ transcription.”

Mice receiving both anti-IL-10R and CpG become severely ill, uniformly moribund between days 6-8 of the experiment.

“however, mice that received both anti-IL-10R and CpG treatments became severely ill, uniformly becoming moribund between days 6-8 of the experiment.”

Anti-IL-10R with CpG causes large amounts of hemophagocytosis in splenic touch preparations and peripheral blood smears.

“Most strikingly, mice treated with anti-IL-10R and CpG developed large amounts of hemophagocytosis, easily seen in both splenic touch preparations and in peripheral blood smears (Figure 8H).”

Mice receiving repeated anti-IL-10R with PBS injections show no phenotype.

“Mice that received repeated anti-IL-10R with PBS injections showed no phenotype;”

Both cDCs and pDCs transcribe IFN-γ in this model.

“This observation is consistent with our finding that both cDCs and pDCs are transcribing IFN-γ in our model.”

The disease process can be ameliorated by IFN-γ blockade even if given after the initial inflammatory stimulus.

“Importantly, the disease process can be ameliorated by IFN-γ blockade, even if given after the initial inflammatory stimulus.”

Disease develops in the absence of hemophagocytes in this model.

“Our model also revealed that disease develops in the absence of hemophagocytes, a cell type often found in cytokine storm syndromes, whose role remains unclear.”

IFN-γ is an important effector cytokine in TLR9-mediated cytokine storm.

“While IFN-γ is clearly an important effector cytokine in TLR9mediated cytokine storm, we were unable to identify a single IFN-γ-producing population that was required for disease.”

Repeatedly CpG-treated mice develop serologic and histologic features of cytokine storm.

“Repeatedly CpG-treated mice develop serologic and histologic features of cytokine storm.”

Pancytopenia occurs in the absence of abundant hemophagocytosis in this model.

“Given that we see pancytopenia in the absence of abundant hemophagocytosis, it is difficult to argue for a role for these cells in the development of these low cells counts.”

Mice treated with neutralizing antibody at day 3 had a reduction of symptoms of the same magnitude as that of mice treated before disease induction.

“Mice treated with neutralizing antibody at day 3 had a reduction of symptoms of the same magnitude as that of mice treated before disease induction.”

Blockade of late-phase IFN-γ is sufficient to ameliorate disease.

“Blockade of late-phase IFN-γ is sufficient to ameliorate disease.”

Hemophagocytosis correlates with more severe disease.

“This result makes a correlation of hemophagocytosis with more severe disease; however, the direction of the causality remains undetermined.”

We report a new model of MAS-like cytokine storm that uses TLR9 stimulation.

“Herein we have reported what we believe to be a new model of MAS-like cytokine storm that uses TLR9 stimulation.”

IFN-γ is required for maximal CpG-induced cytokine storm disease expression.

“IFN-γ is required for maximal CpG-induced cytokine storm disease expression.”

No single IFN-γ-producing population was required for disease.

“While IFN-γ is clearly an important effector cytokine in TLR9mediated cytokine storm, we were unable to identify a single IFN-γ-producing population that was required for disease.”

Removal of B cells, T cells, and NK cells together causes a marked reduction in disease severity.

“However, when B cells, T cells, and NK cells were removed together, a marked reduction in disease severity was seen, suggesting that NK cells and lymphocytes together are critical for maximal disease.”

Repeatedly CpG-treated mice develop clinical features of cytokine storm.

“Repeatedly CpG-treated mice develop clinical features of cytokine storm.”

NK cells are activated by CpG treatment.

“NK cells are activated by CpG treatment.”

A single dose of CpG is not sufficient to induce the MAS-like disease.

“It is reassuring that a single dose of CpG was not sufficient to induce the MAS-like disease.”

Simultaneous deletion of NK cells and lymphocytes partially reduces the severity of CpG-induced HLH/MAS-like syndrome.

“Simultaneous deletion of NK cells and lymphocytes partially reduces the severity of CpG-induced HLH/MAS-like syndrome, whereas cDCs are required for early-phase IFN-γ production but are dispensable for disease.”

Robust hemophagocytosis is not required for the development of other clinical abnormalities associated with MAS initiated by repeated TLR9 stimulation.

“These data suggest that robust hemophagocytosis is not required for the development of other clinical abnormalities associated with MAS initiated by repeated TLR9 stimulation.”

Both CD8α+ cDCs and pDCs are likely contributing to the production of IFN-γ in the repeated CpG treatment model.

“Thus, both CD8α + cDCs and pDCs are likely contributing to the production of IFN-γ in our repeated CpG treatment model, again suggesting a nonlymphoid-derived IFN-γ source.”

Hyperactive innate immune responses can lead to IFN-γ production and the MAS phenotype.

“The data presented in this report support the idea that hyperactive innate immune responses, such as those associated with SJIA, can lead to IFN-γ production and the MAS phenotype.”

• AssumptionRests on an assumption: The CpG-induced MAS-like syndrome in mice is a valid model for human cytokine storm syndromes.

TLR9 agonism is unique in producing this MAS-like syndrome.

“Both our own data with other TLR ligands and previous reports (11) suggest that TLR9 agonism is unique in producing this MASlike syndrome.”

• cites [11] Heikenwalder, 2004, Nat Med. sourceNot checked yet

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• AssumptionRests on an assumption: CpG acts specifically through TLR9 to induce the observed disease.

The mild phenotype of this model is a result of the anti-inflammatory activity of IL-10.

“Interestingly, this mild phenotype is a result of the antiinflammatory activity of IL-10.”

IL-10 is produced partly in response to TLR9-induced IFN-γ.

“This IL-10 is produced partly in response to the TLR9-induced IFN-γ, since CpG-treated mice deficient in IFN-γ make less IL-10 than wild-type mice.”

The paper's data provide insight into MAS-like syndromes in patients with inflammatory diseases in which there is chronic innate immune activation but no genetic defects in cytotoxic cell function.

“Our data provide insight into MAS-like syndromes in patients with inflammatory diseases in which there is chronic innate immune activation but no genetic defects in cytotoxic cell function.”

Absence of CD8+ and NKT cells enhances the repeated TLR9-stimulated phenotype.

“In fact, we found that B2m -/-mice consistently produced more IFN-γ than wild-type mice, suggesting that the absence of CD8 + and NKT cells enhances the repeated TLR9-stimulated phenotype (data not shown).”

NK cell activation (CD69 upregulation) does not require CD8+ T cells.

“The increase in CD69 expression in NK cells was also seen in B2m -/-mice, suggesting that this NK cell activation did not require the presence of CD8 + T cells (Figure 6B).”

NK cells and lymphocytes together are critical for maximal disease.

“However, when B cells, T cells, and NK cells were removed together, a marked reduction in disease severity was seen, suggesting that NK cells and lymphocytes together are critical for maximal disease.”

Patients with SJIA show gene expression profiles consistent with repeated TLR/IL-1 receptor activation.

“Patients with SJIA, another population at risk for MAS, show gene expression profiles consistent with repeated TLR/ IL-1 receptor activation (10).”

• cites [10] sourceNot checked yet

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Inflammatory cells in livers of Rag2-/- and Rag2-/-Il2rg-/- CpG-treated mice are innate immune cells.

“It is also of interest to note that the inflammatory cells seen in the livers of the Rag2 -/-and the Rag2 -/-Il2rg -/- CpG-treated mice must be innate immune cells.”

IL-10 responsiveness can change the degree of pathology seen, including the presence of hemophagocytosis.

“Our results also provide a possible explanation for the spectrum of severity of secondary MAS, in that IL-10 responsiveness can change the degree of pathology seen, including the presence of hemophagocytosis.”

• AssumptionRests on an assumption: The CpG-induced MAS-like syndrome in mice is a valid model for human cytokine storm syndromes.

MAS immunopathology plays a more central role in the disease process of SJIA.

“This observation argues that MAS immunopathology plays a more central role in the disease process of SJIA, as might be predicted by our model of repeated TLR-induced MAS and the data on the gene signatures of TLR activation in these patients.”

• AssumptionRests on an assumption: The CpG-induced MAS-like syndrome in mice is a valid model for human cytokine storm syndromes.

Patients with EBV-HLH have much higher viral loads compared with patients with EBV infectious mononucleosis without HLH.

“Patients with EBV-HLH have much higher viral loads compared with patients with EBV infectious mononucleosis without HLH (33).”

• cites [33] sourceNot checked yet

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Over 40% of patients with MAS/HLH do not have hemophagocytes in bone marrow aspirates.

“Although sampling error is always of concern, it has been reported that over 40% of patients with MAS/HLH do not have hemophagocytes in bone marrow aspirates, suggesting a separation of disease initiation from the development of hemophagocytes (23).”

• cites [23] Gupta, 2008, Pediatr Blood Cancer. sourceNot checked yet

  Not checked in the free run.

In the absence of adaptive immunity, the innate immune system can cause pathogenic inflammation in the MAS disease process.

“Although in an animal with a complete immune system, the hepatic infiltrate is predominately lymphocytic, in the absence of adaptive immunity, we see that the innate immune system can also cause pathogenic inflammation in the MAS disease process.”