Increased TLR4 expression and downstream cytokine production in immunosuppressed adults compared to non-immunosuppressed adults

Abstract

Background: An increasing number of patients have medical conditions with altered host immunity or that require immunosuppressive medications. While immunosuppression is associated with increased risk of infection, the precise effect of immunosuppression on innate immunity is not well understood. We studied monocyte Toll-like receptor (TLR) expression and cytokine production in 137 patients with autoimmune diseases who were maintained on immunosuppressive medications and 419 non-immunosuppressed individuals.

Methodology/principal findings: Human peripheral blood monocytes were assessed for surface expression of TLRs 1, 2, and 4. After incubation with TLR agonists, in vitro production of the cytokines IL-8, TNFalpha, and MIF were measured by ELISA as a measure of TLR signaling efficiency and downstream effector responsiveness. Immunosuppressed patients had significantly higher TLR4 surface expression when compared to non-immunosuppressed adults (TLR4 %-positive 70.12+/-2.28 vs. 61.72+/-2.05, p = 0.0008). IL-8 and TNF-alpha baseline levels did not differ, but were significantly higher in the autoimmune disease group following TLR stimulation. By contrast, baseline MIF levels were elevated in monocytes from immunosuppressed individuals. By multivariable analyses, IL-8 and TNFalpha, but not MIF levels, were associated with the diagnosis of an underlying autoimmune disease. However, only MIF levels were significantly associated with the use of immunosuppressive medications.

Conclusions/significance: Our results reveal that an enhanced innate immune response is a feature of patients with autoimmune diseases treated with immunosuppressive agents. The increased risk for infection evident in this patient group may reflect a dysregulation rather than a simple suppression of innate immunity.

Figure 1. Surface expression of TLRs 1, 2 and 4 in Immunosuppressed adults compared to non-immunosuppressed adults.

TLR surface expression is represented as percentage of CD4-dim cells stained with antibodies to TLR 1, 2, or 4 as assessed by flow cytometery. No statistical difference is seen in TLR 1 or 2 surface expressions in immunosuppressed adults compared to non-immunosuppressed adults. TLR4 surface expression was significantly increased in immunosuppressed adults (70.12±2.28 versus 61.72±2.05, p = 0.0008). NS =  p>0.05; values indicate least squares means from a model adjusted for age group, gender, and race, and bars indicate 1 standard error.

Figure 2. TLR signaling efficiency in Immunosuppressed adults compared to Non-immunosuppressed adults.

Delta IL-8 (Panel A) and Delta TNFα (Panel B) and Delta MIF (Panel C) levels in monocytes (Delta  =  units changed from the baseline unstimulated levels.) TLR ligands were as follows: For TLR1/2, Pam₃CSK₄; 5 µg/ml; for TLR2, LTA; 1 µg/ml; for TLR4, LPS: 0.5 µg/ml, for TLR5 flagellin 2.5 µg/ml. Values indicate least squares means from a model adjusted for age group, gender, and race, and bars indicate 1 standard error.

Figure 3. Effect of underlying disease on cytokine production.

When compared to adults who did not have the diagnosis of RA, SLE or “Diagnosis Other” (all other diseases for which adults were taking immunosuppressant medication), those with RA or “diagnosis other” had significantly higher IL-8 (3A) and TNFα (3B) levels. MIF levels were not significantly associated with the diagnosis of an underlying autoimmune disease (not shown). Values indicate least squares means from a model adjusted for age group, gender, and race and bars indicate 1 standard error.