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Synthetic ligands against TLR2-9 in TruCulture™ - whole blood assays distinguish clinical stages of SIRS (trauma) and sepsis

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Abstract and Figures

To understand the nature of immune dysfunction in patients with sepsis manifesting after severe trauma, a robust whole blood ex vivo test system (TruCultureTM) has been developed. Methods: One milliliter of blood was drawn into separate syringes containing ligands to stimulate the following TLR2-9. Amongst many others, the biomarkers TNF-α, Interleukin-1β and TNF-α followed by IL-1RA and soluble TNF-RII were quantified by multiplexed sandwich immunoassays. The IL-1ratio and TNFratio following stimulation with LPS, were defined as the ratios of IL-1β [pg/ml]/IL-1RA [pg/ml] and TNF-α [pg/ml] /sTNF-RII [pg/ml], respectively. Results: When compared with healthy donors, most TLR induced cytokines were lower in trauma and even lower in sepsis patients’ cultures. An exception was the TLR2 stimulation, which induced more inflammatory and anti-inflammatory cytokines as well as soluble receptors in trauma and sepsis than in healthy donors. Among the other TLR responses, TLR3 was most dramatically downregulated in patients with trauma and even more in sepsis patients. Calculating the IL-1ratio and the TNFratio, we found a patient-type specific ratio of ligand to antagonists. Healthy donors had a median IL-1ratio of 1.48 and a median TNFratio of 2.73, trauma patients had 10times -, and sepsis patients had 100times lower IL-1 and TNFratios. Conclusion: The here developed TruCultureTM ex-vivo whole blood TLR stimulation test is valid to correlate a defined response pattern to the clinically established stages trauma and sepsis. Results may substantially contribute to signalling pathways leading to immune dysfunction.
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+LW[(UHLZ[OLZPVSVN`:[LPUOVL]LSZ[Y " <ST.LYTHU`"
9)4+\]HS9VHK;L_HZ 
To understand the nature of immune dysfunction in patients with sepsis mani-
festing after severe trauma, a robust whole blood ex vivo test system (TruCul-
) has been developed.Methods: One milliliter of blood was drawn into sepa-
rate syringes containing ligands to stimulate the following TLR2-9. Amongst many
1ratio and TNFratio 
] /sTNF-RII[pg/Ml], respectively.
Results: When compared with healthy donors, most TLR induced cytokines were
lower in trauma and even lower in sepsis patients’ cultures. An exception was the
        -
tokines as well as soluble receptors in trauma and sepsis than in healthy donors.
Among the other TLR responses, TLR3 was most dramatically downregulated in
patients with trauma and even more in sepsis patients. Calculating the IL1ratio
and the TNFratio
Healthy donors had a median IL1ratio of 1.48 and a median TNFratio of 2.73,
trauma patients had 10times -, and sepsis patients had 100times lower IL1 and
TNFratios. Conclusion: The here developed TruCulture
ex-vivo whole blood
    -
*VKL!  
 4VUK\aaP,KP[VYL c 7YVJLLKPUNZ
cally established stages trauma and sepsis. Results may substantially contribute to
signalling pathways leading to immune dysfunction.
    
and hypotension, and may impair organ functions. The severe form of an initial insult
           -
scribes SIRS followed by the presence of immunologically uncontrolled pathogens
or their toxins[1]. Many clinical parameters have been measured, and, also, several
potential markers have been suggested to describe the transitions during the disease
course[2-4]. Combining clinical outcome, proteomics as the important element of a
system biology approach enables an integrative outlook of a complex nature of signal-
ing networks in sepsis and may help identify critical regulatory nodes for therapeutic
manipulation[5,6]. One approach is to restimulate patients’ blood cells via their Toll-
like-receptors (TLR) representing the pattern recognition receptors of the immune
system. TLR ligands constitute the most important molecular structures called PAMPs
(pathogen-associated molecular patterns) (PAMPS)[7,8].
Sixteen ICU postoperative/posttraumatic trauma (SIRS) or sepsis patients were
included in this prospective pilot study and recorded daily over 4–15 days. From
admission to ICU to discharge from ICU, the assessment of clinical data was based
cal variables, age, type of admission (medical and scheduled/unscheduled surgery),
cancer and hematologic malignancy) [9] for severity of disease, and SOFA (Se-
quential Organ Failure Assessment) Score (status of 6 organ systems (respiration,
coagulation, liver, cardiovascular, central nervous system, renal)[9] for severity of
organ dysfunctions.
years.Male to female ratio was 4/5. The median SAPSII was 25 (range 16 to 32).
The median SOFA was 5 (range 1 to 15).
          
          
to female ration was 6/1. The median age was 58 years (range 42-76 years). The
median SAPSII was 36 (range: 15 to 43). The median SOFA was 5 (range 0 to 8).
ranged from 18 – 53.
4HYJO [O[O4\UPJO.LYTHU`
The TruCulture
system ( isdesigned to draw
one milliliter of blood was drawn into separate syringes each of which contained
just one ligand to stimulate the following TLRs: TLR1/2(MALP); -2/6(Pam3Cys),-
The biomarkers secreted by blood cells as well as soluble receptors released from
     
( The parameters IL1ratio and o following
 
GraphPadPrism™ 5 was used to create graphs and calculate differences between
groups using Mann-Whitney-U tests.
      -
dardized assay (TruCulture
       -
duced by trauma alone or associated with a major infectious complication (sepsis or
septic shock) alters the capacity of the immune system to respond to an additional
Figure 1 shows that blood sampling into TruCulture
tubes is easy and can be
accomplished in less than 90 seconds per patient. Supernatants can be harvested in
a bedside fashion and may be shipped for multiplexed determination of biomarker
concentrations to an adequate research laboratory.
Figure 2 shows the spontaneous release of IL-1ß, TNF-α, IL-1RA and sTNF-RII
determined in culture supernatants w/o stimulant. With the exception of the shock
           
sTNF-RII. One may interprete in vivo activation by the analysis of spontaneous
α and its binding molecules, the higher
release of antagonists in sepsis patients may be explained by the (compensatory)
         
been further elaborated by others [1,10-12].
  
Ex Vivo Simulation of Infection Using TLR- Whole Blood Stimulation (ICU)
IL-1ß w/o stimulus
Healthy Trauma Sepsis
IL-1RA w/o stimulus
Healthy Trauma Sepsis
TNF-D w/o stimulus
Healthy Trauma Sepsis
sTNF-RII w/o stimulus
Healthy Trauma Sepsis
Fig 1 - 
        !"  #$% 
#&'%() *
Fig 2 - +,!$-./$0,!$"1./$",,
 %
4HYJO [O[O4\UPJO.LYTHU`
were recorded and are summarized in Figure 3. When compared to healthy donors,
lowing TLR2/6 (Pam3Cys) and TLR2/1 (FSL-1) stimulation (Figure 3). However,
          -
stimulation by ligands binding to TLR4 has been described previously[13]. This
context may explain why sepsis patients’ blood cells have a reduced response to
            
increased TLR2 responsiveness as observed in our trauma patient population has
           -
         
cytokines as well as endotoxin to upregulate the TLR2 expression and its response.
The generally lower cytokine responsiveness of patients with sepsis/septic shock
following TLR3, -4, and -5 stimulation is not explained by a lower surface expres-
sion, since TLR3 is an endosomally expressed receptor and its response requires li-
mechanism explaining multi-receptor targeted tolerance[16]. These results suggest
follow a systematic response pattern in trauma and sepsis patients. Although TLR9
in sepsis blood cells as compared to trauma and healthy controls. The same is true
whole blood stimulation assays[17] and may explain this difference.
Since van Endert recently described that endosomal TLR9 requires proteolytic
activation by AEP[18] it may be worth investigating whether maturation of TLR9 in
patients with sepsis/septic shock differs from the maturation status in trauma or sep-
sis. The difference between ODN2216 and ODN2006 representing type A and type
B oligonucleotides can be also explained by the recently described co-stimulatory
function of high mobility gene box-1 (HMGB-1)[19]. HMGB-1 further binds to
soluble RAGE (receptor for advanced glycation end products) and this complex is
In addition to the increased TLR9 responsiveness, sepsis patients’ blood cells
also true for ligands binding to TLR2, -3, -4, -5, and -7. This effect further supports
the concept of immune tolerance in this patients population[11].
 
danger-receptor complex [22], this molecule is in part responsive for elevations of
alone restricted to infectious complications[22]. Similarly, soluble TNF-RII binds
        
has been shown to be restricted to pre-treatment models[23] [24].
1 10 100 1000 10000 100000
Poly I:C
ODN22 16
ODN20 06
no TLR-Ligand
IL-1ß concentration in pg/ml
1 10 100 1000 10000 100000
Poly I:C
ODN22 1 6
ODN20 0 6
no TLR-Ligand
TNF-D concentration in pg/ml
1 10 100 1000 10000 100000
Poly I:C
ODN22 16
ODN20 06
no TLR-Ligand
IL-1ß concentration in pg/ml
1 10 100 1000 10000 100000
Poly I:C
no TLR-Ligand
TNF-D concentration in pg/ml
1 10 100 1000 10000 100000
Poly I:C
no TLR-Ligand
IL-1ß concentration in pg/ml
1 10 100 1000 10000 100000
Poly I: C
no TLR-Ligand
TNF-D concentration in pg/ml
Fig 3 - !" 
   2( 
4HYJO [O[O4\UPJO.LYTHU`
As summarized in Table 1a, the median ratio calculated of the amount of
tion is 1.48 in healthy donors and declines to a median value of 0.18 in trauma
      TNFratio, is 2.37 in healthy donors,
0.43 in trauma patients and 0.06 in sepsis patients. We conclude that these ratios
also calculated the respective IL-1ratio and TNFratio following other TLR-spe-
        
(data not shown).
Table 1b shows an increased release of sTNF-RII and IL-1RA following TLR4
          
secretion is lower in SIRS and even less in sepsis cultures. Table 2 clearly shows
their antagonists (IL-1RA, sTNF-RII)between healthy volunteers and SIRS/Sep-
sis patients. However, the amount of IL-1RA is not remarkably different between
         
           
Healthy Trauma Sepsis
median (range) median (range) median (range)
Table 1b
Trauma Sepsis
median (range) median (range) median (range)
IL-1ȕ>QJPO@ 25.5 11.2-53.8 5.2 0.0-15.3 2.8 0.3-5.0
IL-1RA [ng/ml] 11.4 8.8-18.3 23.0 8.9-39.5 26.1 4.2-54.6
TNF-Į [ng/ml] 12.2 6.0-17.8 4.54 0.2-8.4 1.9 0.3-2.7
sTNF-RII [ng/ml] 5.2 2.1-7.5 11.0 7.0-17.0 30.0 6.8-45.0
Tab. 1 a and 1 b - +,!$-,!$"1./$0./$",,!"%
 
   !"%*
Tab. 1 a
Tab. 1 b
heterogeneity in the sepsis group, including cases with severe sepsis as well as sep-
tic shock. A larger study may clarify this issue.
The biomarker results presented in this pilot study supports a number of gene
er, are laborious expensive and time consuminghand and, considerably differ in
the expressed gene signature due to underlying diseases [25-27] and experimental
models[27]. The proteomic approach using TruCulture
stimulation is suitable for patients undergoing different stages of a disease as well as
different diseases leading to the manifestation of severe SIRS and sepsis. The em-
well as oxidative stress by PAMP as major effectors in SIRS and sepsis.
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Healthy vs SIRS
Healthy vs.
SIRS vs. Sepsis
TNF-Į 0.012 0.0025 0.142
sTNF-RII 0.002 0.0101 0.055
IL-1ȕ 0.004 0.0025 0.211
IL-1RA 0.060 0.2677 0.760
0,003 0,006 0,091
0,001 0,003 0,042
Tab. 2 - +45$) ./$0./$",,,!$-,!$"1
 ,!$./*
4HYJO [O[O4\UPJO.LYTHU`
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erance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase. J Immunol
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munol 2000:165(10):5767-5772.
16. Lehner MD, Morath S, Michelsen KS, et al. Induction of cross-tolerance by lipopolysaccharide and
tors. J Immunol 2001:166(8):5161-5167.
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loop system: mechanisms and immunomodulatory effects. J Immunol 2009:183(10):6724-6732.
18. van Endert P. Toll-like receptor 9: AEP takes control. Immunity 2009:31(5):696-698.
19. Tian J, Avalos AM, Mao SY, et al. Toll-like receptor 9-dependent activation by DNA-containing
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say and comparison with standard enzyme-linked immunoassay for cytokine analysis. Shock
            
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... 8 The immunotoxic effect of increasingly relevant doses of AFB 1 on 1 class of pivotal immune surveillance molecules, pattern recognition receptors (PRRs), is still unclear. The vital role of PRR in inflammation to clear microbial infection [9][10][11] is well documented. Dendritic cells (DC) are key antigenpresenting cells (APCs) and professionally link innate and acquired immunity. ...
... The roles of TLR in immune regulation, immune-and inflammationmediated diseases, sepsis, autoimmune disorders, allergy, cancer, and many other infectious/noninfectious diseases remain the focus. 9,10,11,20,22,[34][35][36] Although continuous efforts have been made to consume AFB 1 -free feeds and foods, 20,37 unfortunately, humans and especially farm animals are still inevitably exposed 21 ; this is more frequent in developing countries. 1,38 Although it is believed that the exposure of food-producing animals and humans to AFB 1 is relatively low in Europe, [23][24][25] but occupational exposure to AFB 1 is alarming, especially for indoor farmers/workers, whose exposure is by both the inhalation and the oral routes as well as healthy skin; as such, the selected level of AFB 1 is increasingly reasonable, even in Europeans, 26 unquestionably making the dose rationale in our study relevant. ...
Full-text available
Aflatoxins (AFs) are highly hazardous fungal biometabolites usually present in feeds and foods. Aflatoxin B1 (AFB1) is the most toxic and a known carcinogen. Toll-like receptors (TLRs), highly expressed by myeloid dendritic cells (DC), are key innate immune-surveillance molecules. Toll-like receptors not only sense pathogen-associated molecular patterns but also contribute to infections and cancer. To assess AFB1-TLR interactions on human myeloid DC, pure CD11c(+) DC were generated from monocytes isolated from healthy individuals and then exposed to relevant level of AFB1 for 2 hours. Both quantitative polymerase chain reaction and flow cytometric assays were used to quantify, respectively, expression of TLR2 and TLR4 at the messenger RNA (mRNA) and protein levels in these DC. Levels of interleukin (IL) 1β, IL-6, and IL-10 were also analyzed in AFB1- and mock-treated DC. Compared to nontreated CD11c(+) DC, expression levels of both TLR2 and TLR4 mRNA and proteins were significantly upregulated in AFB1-treated cells. Further, although IL-10 levels in AFB1-treated DC were similar to those in the mock-treated DC, the AFB1-exposed DC secreted higher amounts of IL-1β and IL-6. Dendritic cells are sensitive to environmentally relevant level of AFB1, and TLR2 and TLR4 are involved in sensing AFB1. Considering the broad roles of TLR2, TLR4, and DC in immunity and infections, our novel findings open a new door to understanding the molecular mechanisms and functional consequences of AFB1 in inducing immunodysregulation, immunotoxicity, and thus (non)infectious diseases in humans.
... A static cell culture model meeting all of these criteria was developed more than two decades ago for testing drug activities in clinical studies (TruCulture®, (19,20)). It is generally used as a static cell culture system and was primarily developed for ex vivo testing of pharmaceutical drug effects on the immune system. ...
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Background Common in vitro cell culture systems for testing implant material immune-compatibility either employ immortal human leukocyte cell lines or use isolated primary cells. Compared to in vivo conditions, this generates an environment of substantially reduced complexity, often lacking important immune cell types, such as neutrophil granulocytes and others. This paper describes an innovative human whole blood culture model for in vitro testing of implant materials under in vivo-like conditions. The major goal of this culture model was to maintain as much of the naturally inherent complexity of immune cell interactions as possible and to avoid errors often caused by stressful conditions during cell preparation. Methods A closed, CO2-independent, tube-based culture vessel was used, containing one milliliter of freshly drawn human blood for each sample. The cultures were occasionally rotated to increase immune cell contacts with the test materials. Immune cell responses were examined by multiplexed cytokine analysis. Results Three different types of commercially available implant materials i.e. barrier membranes, used for dental, trauma and maxillofacial surgery, were examined for their potential interactions with immune cells. The barrier membranes were either of synthetic (i.e. the polymers polytetrafluoroethylene, PTFE, and polycaprolactone, PCL), or of natural origin (porcine collagen membrane). The results identified characteristic differences in the overall activity profiles with very low immune cell responses for PTFE, intermediate ones for collagen, and strong reactions towards PCL. Conclusions This innovative human whole blood in vitro model, using a complex, organotypic matrix and all immune cells available in peripheral blood, is an excellent, easy to standardize tool to categorize immune cell responses to implant materials. Compared to in vitro cell culture systems used for material research, this new assay system provides a far more detailed picture of response patterns the immune system is able to develop when interacting with different types of materials and surfaces.
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To investigate interactions between the endothelium and leukocytes in patients with sepsis, we measured soluble adhesion molecules (sE-selectin and sICAM-1), von Willebrand factor antigen (vWf:Ag), myeloperoxidase (MPO), and lactoferrin (Lacto-f) as plasma markers of endothelial and neutrophil activation. We tested whether the five proteins were predictors of clinical severity, which was evaluated by simplified acute physiological score (SAPS), number of organ failures (MOF), acute lung injury (ALI), and subsequent final outcome. Levels of the five plasma markers were higher in patients with severe infection (n = 25) than in patients without sepsis (n = 7) and healthy volunteers (n = 9). In the study population, levels of sE-selectin, sICAM-1, and vWf:Ag were higher for nonsurvivors as well as for patients with septic shock or with bacteremia, and they were correlated with SAPS and MOF. Survival outcome was predicted with high sensitivity and specificity by initial plasma levels of sICAM-1 and vWf:Ag. The initial sICAM-1 level appeared to be an independent prognostic variable, based on a logistic regression analysis. Unlike sE-selectin, sICAM-1 remained at high levels indefinitely in nonsurvivors. We conclude that, unlike neutrophil activation markers, levels of endothelium-derived soluble adhesion molecules and vWf:Ag in severe sepsis syndrome are correlated with the severity of illness and may be considered as predictors of survival outcome.
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In this study, we report on initial efforts to discover putative biomarkers for differential diagnosis of a systemic inflammatory response syndrome (SIRS) versus sepsis; and different stages of sepsis. In addition, we also investigated whether there are proteins that can discriminate between patients who survived sepsis from those who did not. Our study group consisted of 16 patients, of which 6 died and 10 survived. We daily measured 28 plasma proteins, for the whole stay of the patients in the ICU. We observed that metalloproteinases and sE-selectin play a role in the distinction between SIRS and sepsis, and that IL-1alpha, IP-10, sTNF-R2 and sFas appear to be indicative for the progression from sepsis to septic shock. A combined measurement of MMP-3, -10, IL-1alpha, IP-10, sIL-2R, sFas, sTNF-R1, sRAGE, GM-CSF, IL-1beta and Eotaxin allows for a good separation of patients that survived from those that died (mortality prediction with a sensitivity of 79% and specificity of 86%). Correlation analysis suggests a novel interaction between IL-1alpha and IP-10. The marker panel is ready to be verified in a validation study with or without therapeutic intervention.
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Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.
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Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.
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Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.
Toll-like receptor 9 (TLR9) requires proteolytic maturation to acquire signaling capacity; however, the involved protease(s) is unclear. In this issue of Immunity, Sepulveda et al. (2009) demonstrate that in dendritic cells, asparaginyl endopeptidase is a key protease that controls TLR9 maturation.
An intact innate and acquired immune response are essential for defeating systemic microbial infections. Recognition molecules, inflammatory cells, and the cytokines they produce are the principal means for host tissues to recognize invading microbes and to initiate intercellular communication between the innate and acquired immune systems. However, activation of host innate immunity may also occur in the absence of microbial recognition, through expression of internal "danger" signals produced by tissue ischemia and necrosis. When activation of the innate immune system is severe enough, the host response itself can propel the patient into a systemic inflammatory response syndrome (SIRS), or even multiple system organ failure (MSOF) and shock. Although most patients survive the initial SIRS insult, these patients remain at increased risk of developing secondary or opportunistic infections because of the frequent onset of a compensatory anti-inflammatory response syndrome (CARS). The initial activation of the innate immune response often leads to macrophage deactivation, T-cell anergy, and the rapid apoptotic loss of lymphoid tissues, which all contribute to the development of this CARS syndrome and its associated morbidity and mortality. Initial efforts to treat the septic patient with anticytokine therapies directed at the SIRS response have been disappointing, and therapeutic efforts to modify the immune response during sepsis syndromes will require a more thorough understanding of the innate and acquired immune responses and the increased apoptosis in the lymphoid tissue.