Investigation of the differential potentials of TLR agonists to elicit uveitis in mice

Abstract

TLRs are critical for host defense and innate immunity. Emerging evidence also supports a role for TLRs in many chronic inflammatory diseases, including inflammatory eye disease, known as uveitis. The activation of TLR4 by endotoxin induces a standard model of murine uveitis. How activation of additional TLRs influences the onset and/or severity of anterior uveitis has not been examined. We sought to elucidate the potential of TLRs (TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7/8, and TLR9) to trigger uveitis in mice. Directly stimulated iris/ciliary body explants demonstrated a marked increase in production of inflammatory cytokines TNF-α, IL-6, IP-10/CXCL10, MCP-1, and KC with relatively little production of IFN-γ, IL-10, IL-12p40, IL-12p70, IL-17, IL-1β, IL-4, or RANTES. The cytokine-response profiles were comparable amongst the TLR agonists, albeit some differences were noted, such as greater IP-10 production following TLR3 activation. Intra-ocular injection of TLR agonists increased leukocyte interactions with the endothelium of the iris vasculature and resulted in chemotaxis into the iris tissue. Assessment of leukocytic responses by ivt videomicroscopy and histology revealed quantitative differences amongst responses to the TLR agonists with respect to the timing and numbers of rolling, adhering, iris-infiltrating, and aqueous humor-infiltrating cells, along with cytokine levels in vivo. Our data demonstrate the eye's responsiveness to a diverse array of microbial products, which activates TLRs, and reveal differences in relative cellular response among the various TLR agonists in vivo.

Figure 1.. TLR stimulation of the iris/ciliary body ex vivo results in unique profiles of cytokine production.

Organotypic iris/ciliary body cultures were stimulated with 10 μg/ml of the indicated TLR agonists. Cytokine levels in the culture supernatants were determined by multiplex ELISA (Luminex) at 24 h poststimulation. Data are shown as mean ± sem of combined values from three independent experiments with three mice each. *P < 0.05 comparison between media control and TLR agonist-stimulated samples.

Figure 2.. Activation of TLR4 by lipid A.

The numbers of rolling, adhering, and infiltrating cells within the iris vasculature and tissue as a function of time in response to 250 ng lipid A were enumerated with ivt videomicroscopy. Data are shown as mean ± sem of combined values from two independent experiments. *P < 0.05 comparison between lipid A-injected eyes and saline-injected control eyes (n=9–15 mice/treatment/time-point).

Figure 3.. Activation of TLR2/1 and TLR2/6 heterodimers by Pam3CSK4 and FSL-1.

The numbers of rolling, adhering, and infiltrating cells within the iris vasculature and tissue as a function of time in response to 1 μg Pam3CSK4 (A) or 0.4 μg FSL-1(B) were enumerated with ivt videomicroscopy. Data are shown as mean ± sem of combined values from two independently performed experiments. *P < 0.05 comparison between Pam3CSK4- or FSL-1-injected eyes and saline-injected control eyes (n=9–20 mice/treatment/time-point).

Figure 4.. Activation of TLR5 by flagellin.

The numbers of rolling, adhering, and infiltrating cells within the iris vasculature and tissue as a function of time in response to 0.4 μg flagellin were enumerated with ivt videomicroscopy. Data are shown as mean ± sem of combined values from two independently performed experiments. *P < 0.05 comparison between poly (I:C)-injected eyes and saline-injected control eyes (n=9–15 mice/treatment/time-point).

Figure 5.. Uveitic response to nucleic acids that trigger TLR3, -7, -8, or -9.

The numbers of rolling, adhering, and infiltrating cells within the iris vasculature and tissue as a function of time in response to 4 μg poly (I:C) (A), 4 μg R848 (B), or 6.5 μg CpG (C) were enumerated by ivt videomicroscopy. Data are shown as mean ± sem of combined values from two independently performed experiments. *P < 0.05 comparison between TLR agonist-injected eyes and saline-injected control eyes (n=9–15 mice/treatment/time-point).

Figure 6.. Histological assessment of the impact of TLR activation on inflammation within the anterior segment.

Representative photos (original magnifications, 400×) of tissue sections of the eyes from mice injected with each of the TLR agonists and assessed 6 h postinjection by H&E staining. The numbers of cells within the aqueous humor of the anterior segments are graphically depicted (lower right panel). Data are shown as mean ± sem. *P < 0.05 comparison between TLR agonist-injected eyes and saline-injected control eyes (n=6 mice/treatment).

Figure 7.. Evaluation of cytokine responses to TLR agonists in vivo.

Whole eye-tissue homogenates were evaluated for the indicated levels of cytokines by multiplex ELISA at 6 h postinjection. Data are shown as mean ± sem (n=8 mice/treatment). *P < 0.05 comparison between ivt saline controls and TLR agonist-injected mice.