We analyzed TLR responsiveness in PBMNCs from seropositive individuals and compared it with seronegative subjects. We document for the first time that freshly isolated monocytes and mDCs from autoantibody-positive subjects have dysregulated TLR-induced IL-1β and IL-6 pathways compared with autoantibody-negative individuals via mechanisms that may be linked with age but not with altered proportions of peripheral monocytes and DCs, aberrant levels of HbA1c, or the presence of one versus more than one autoantibody. We also document that autoantibody-positive subjects have lower levels of CXCL-10 in their blood. The observation that altered innate immunity is detectable in at-risk subjects before disease onset supports the hypothesis that this alteration may be linked with early disease mechanisms.
Our findings that seropositive subjects have altered IL-1β and IL-6 pathways are compatible with our recent finding that newly diagnosed patients with T1D have higher proportions of ex vivo monocytes secreting IL-1β and elevated numbers of TLR-induced IL-1β−expressing monocytes compared with healthy subjects (11
). Our data lend support to the hypothesis that changes in TLR responsiveness are detectable in monocytes and mDCs from humans with ongoing autoimmunity before and after disease onset. In addition, our results are also in accordance with a previous report that patients with T1D have higher frequencies of ex vivo IL-1β–expressing monocytes determined using ELISPOT (21
). It was proposed that monocytes could be involved in T1D onset by inducing an IL-17 response (21
Observations from a number of experimental systems have suggested the potential involvement of the IL-1 pathway in islet destruction. We recently demonstrated that the IL-1 pathway is involved in the proinflammatory response leading to virus-induced T1D (20
). IL-1, alone or combined with other proinflammatory cytokines such as IFN-β, can cause β-cell destruction in islets from humans and animals and perfused pancreas via pathways involving mitogen-activated protein kinase and nuclear factor (NF)-κB (23
). Blocking the IL-1 pathway protected from T1D in animal models and treating non–diabetes-prone animals with IL-1 caused transient insulinopenic diabetes (25
). Finally, earlier reports have linked IL-1 cytokine family members, including IL-1β, IL-1R1, and IL-1R2, with human T1D (26
Our finding that subjects at risk for T1D have reduced TLR-induced monocyte expression of IL-6 is reminiscent of our previous observation that PBMNCs from new-onset patients activated with LPS contain lower frequencies of IL-6–expressing mDCs (11
). Others have also reported a reduced IL-6 response in monocytes from patients with T1D (27
). IL-6 promotes inflammation (28
) and adaptive immune responses (29
); however, it also suppresses the functions of various cell subsets, including macrophages and synovial fibroblasts. IL-6 induces anti-inflammatory factors, such as IL-1 receptor antagonist and glucocorticoids, inhibits the production of proinflammatory cytokines, such as IL-1, tumor necrosis factor, and IL-12, and downregulates the expression of adhesion molecules (30
). Thus, reduced IL-6 responses in seropositive individuals could potentially be part of early mechanisms leading to loss of immune regulation and consequently T1D.
It is currently unclear how dysregulated TLR-induced IL-1β and IL-6 responses in monocytes and mDCs are involved in mechanisms leading to islet destruction. There is also uncertainty regarding the mechanism underlying this alteration. The data demonstrating that subjects with more than one autoantibody have similar TLR-induced IL-1β and IL-6 responses as subjects with only one autoantibody is interesting given the large difference in the risk for T1D between these groups (5
). One potential explanation is that the increased TLR responses seen in seropositive subjects may be linked with mechanisms triggering anti-islet autoimmunity rather than progression to T1D. The interpretation of these data should be constrained by the caveat that most of the seropositive subjects included in this study expressed only one or two autoantibodies. It remains to be determined whether monocytes and mDCs from individuals with three autoantibodies react differently to TLR ligation compared with subjects with one and two autoantibodies. Also intriguing is the observation that altered IL-1β responses are more prevalent in young children (<11 years) compared with IL-6 responses that appeared to be more readily detectable in adolescents. It is tempting to speculate that the altered innate immune pathways detectable in seropositive individuals could be occurring at different ages or disease stages.
The altered ability of monocytes and mDCs to respond to TLR ligation could be involved in disease mechanisms by altering the balance between proinflammatory and regulatory mechanisms in the periphery and/or the pancreatic islets. This could result in greater islet inflammation and anti-islets T-cell upregulation (31
). Dysregulated TLR-induced cytokine and chemokine responses could be induced, at least in part, by genes linked with mediating innate immune functions or innate regulatory mechanisms. Our data suggest that the mechanism of altered TLR-induced IL-1β and IL-6 responses in seropositive subjects does not involve altered TLR-induced NF-κB phosphorylation or TLR expression (data not shown). It is important to note, however, that although all study participants were free of hyperglycemia, we are unable to exclude the possibility that the altered innate immunity we observed in these subjects is in fact a result of subtle changes in glucose metabolism triggered by ongoing proinflammatory responses in pancreatic islets (32
In contrast to our previously published data documenting elevated levels of IFN-γ, IL-1β, and CXCL-10 in sera from new-onset patients (11
), we observed that serum expression levels of CXCL-10 were lower in seropositive compared with seronegative subjects, whereas the amount of IFN-γ was similar in these subject groups. Why seropositive individuals have reduced amounts of CXCL-10 in their blood is unknown. This reduction could be a consequence of recruitment of chemokine-secreting monocytes, DCs, or B lymphocytes from the peripheral blood to inflamed tissues. The observation that the level of serum CXCL-10 is reduced in subjects with more than one versus only one autoantibody is compatible with this hypothesis, as it would be reasonable to hypothesize that individuals with more than one autoantibody may have a higher degree of inflammation, resulting in the recruitment of greater numbers of CXCL-10–producing cells to inflamed islets. The possibility that CXCL-10 is directly involved in the inflammation leading to T1D is supported by a number of reports. CXCL-10 is detectable in human pancreatic islets from patients with new-onset T1D (34
) and can also be found in pancreatic lymph nodes and islets from animal models of virus-induced T1D (35
; our unpublished data). Our opposing observations indicating an increase versus no change or a decrease in proinflammatory cytokine and chemokine levels in new-onset patients versus prediabetic individuals, respectively (current report; 11
), could imply that alterations detected in subjects with new-onset T1D may be at least partly linked with late stages of T1D or hyperglycemia and inflammation previously documented in patients with T1D (32
In summary, our data demonstrate that individuals with active anti-islet autoimmunity have altered TLR-induced IL-1β– and IL-6–signaling pathways before disease onset. Future longitudinal studies in prediabetic individuals progressing to T1D will be required to evaluate the potential link between the innate immune system and disease onset. Studies to identify early immune pathways involved in T1D are critical not only for designing anti-inflammatory therapeutic interventions but also for identifying biomarkers that could be used for monitoring inflammation and disease progression.