Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea

Abstract

Sterile inflammation underlies many diseases of the cornea including serious chemical burns and the common dry eye syndrome. In search for therapeutic targets for corneal inflammation, we defined the kinetics of neutrophil infiltration in a model of sterile injury to the cornea and identified molecular and cellular mechanisms triggering inflammatory responses. Neutrophil infiltration occurred in two phases: a small initial phase (Phase I) that began within 15 min after injury, and a larger second phase (Phase II) that peaked at 24-48 h. Temporal analysis suggested that the neuropeptide secretoneurin initiated Phase I without involvement of resident macrophages. Phase II was initiated by the small heat shock protein HSPB4 that was released from injured keratocytes and that activated resident macrophages via the TLR2/NF-κB pathway. The Phase II inflammation was responsible for vision-threatening opacity and was markedly suppressed by different means of inhibition of the HSPB4/TLR2/NF-κB axis: in mice lacking HSPB4 or TLR2, by antibodies to HSPB4 or by TNF-α stimulated gene/protein 6 that CD44-dependently inhibits the TLR2/NF-κB pathway. Therefore, our data identified the HSPB4/TLR2/NF-κB axis in macrophages as an effective target for therapy of corneal inflammation.

Figure 1. Time course of cellular and molecular changes in the rat cornea after sterile injury

1. Time course of neutrophil infiltration in the cornea as measured by MPO. Neutrophils infiltrated the cornea in two phases: Phase I, a small initial phase that began within 15 min and reached a plateau level at 4–8 h; Phase II, a larger infiltration of neutrophils that followed and peaked at 24–48 h. Then neutrophils gradually decreased over 7 days that reflected the recovery phase. n = 5 at each time-point.

2. Microarray data of the cornea at 0, 4 and 24 h after sterile injury. The up-regulated genes were classified into three groups: Group A, genes that were up-regulated at 4 h and returned towards normal at 24 h; Group B, genes that were up-regulated at 4 h and remained steady at 24 h; Group C, genes that were gradually up-regulated over 24 h in parallel with neutrophil infiltration. n = 4 at each time-point.

3. Real-time RT PCR assays of representative genes in Groups A, B and C. n = 5 per group at each time-point. Error bars represent means ± s.e.m.

Figure 2. Increased expression of SN and HSPB4 in the rat cornea and keratocytes after sterile injury

A,B. Western blots of SN and HSPB4 in the cornea. SN was immediately released into the cornea after injury. HSPB4 rapidly increased after injury and reached a peak at 4 h. C. SN was increased in the blood of injured rats in the same temporal sequence as its expression in the cornea. n = 5 at each time-point. Error bars represent means ± s.e.m. D. HSPB4 increased rapidly in the cornea after injury with a peak at 4 h. n = 5 at each time-point. Error bars represent means ± s.e.m. E. HSPB4 was released from injured cornea to the media during incubation in culture. n = 5 per group at each time-point. Error bars represent means ± s.e.m. F,G. Immunohistochemistry of the cornea after injury demonstrated increases of SN within 2 h and increases of HSPB4 within 4 h. H,I. Real-time RT PCR assays of selected Group A genes (neuropeptides, small HSPs and crystallins) in the cornea. n = 5 per group at each time-point. Error bars represent means + s.e.m. J. Real-time RT PCR assays of selected Group A genes (small HSPs and crystallins) in keratocytes incubated with extracts of necrotic cornea. n = 5 per group. Error bars represent means + s.e.m. K. Assays for aconitase activity in the injured cornea as a measure for ROS. n = 5 at each time-point. Error bars represent means ± s.e.m. L. Keratocytes expressed HSPB4 in response to incubation with H₂O₂ to generate ROS. n = 5 per group. Error bars represent means + s.e.m. HSPB.

Figure 3. SN reproduced the Phase I inflammatory response and HSPB4 reproduced Phase II

1. The injection of the recombinant SN (0.2 ng) into the corneal stroma of rats reproduced Phase I, but not Phase II. n = 5 per group at each time-point. p values, versus control (PBS-treated). Error bars represent means ± s.e.m.

2. Injection of the recombinant HSPB4 (100 ng) into the corneal stroma reproduced Phase I and Phase II. n = 5 per group at each time point. p values, versus control (PBS-treated). Error bars represent means ± s.e.m.

3. The corneal opacity developed around the injection site (arrows) of HSPB4 (100 ng) 24 h after injection (top panels). Sections were stained with H&E or immunostained for neutrophil elastase. Increased neutrophil infiltration was observed around the site of HSPB4 injection (arrows) (middle and bottom panels).

4. Topical administration of a calcium channel blocker (Diltiazem) 15 min prior to injury significantly decreased Phase I neutrophil infiltration in the cornea after sterile injury. n = 5 per group. Error bars represent means + s.e.m.

5. Subconjunctival injection of either polyclonal (pAb) or monoclonal (mAb) antibodies to HSPB4 significantly decreased Phase II neutrophil infiltration in the cornea after sterile injury. n = 5 per group. Error bars represent means + s.e.m.

6. HSPB4-knockout mice (Hspb4^−/−) developed significantly less Phase II inflammatory response in the cornea after sterile injury than wild-type control mice (129S6/SvEvTac). n = 8 per group. Error bars represent means + s.e.m.

Figure 4. Sterile injury-induced inflammation was markedly decreased in the cornea after depletion of resident macrophages

A-C. Sterile injury was made to the rat cornea after resident macrophages were depleted by subconjunctival injections of clodronate-encapsulated liposome (Cl₂MDP-LIP) on day −2 (2 days before injury) and day 0 (immediately after injury). The cornea was evaluated for neutrophil infiltration by assays for MPO (A), H&E staining (B) and immunostaining for neutrophil elastase to identify neutrophils (C). (A) Neutrophil infiltration measured by MPO was markedly decreased in the cornea 24 h after injury by injection with clodronate-encapsulated liposome, compared to PBS-encapsulated liposome-injected controls (PBS-LIP). n = 5 per group at each time-point. p values, versus control (PBS-LIP-treated). Infiltration of inflammatory cells (B) and neutrophils (C) was also markedly decreased in the macrophage-depleted cornea. Error bars represent means + s.e.m.

Figure 5. HSPB4 induced the Phase II inflammatory response by activating resident macrophages through TLR2/NF-κB signalling

A. Intrastromal injection of HSPB4 (100 ng) did not induce the Phase II response when corneal macrophages were depleted by subconjunctival injection of clodronate-encapsulated liposome (Cl₂MDP-LIP). n = 5 per group at each time point. p values, versus control (PBS-LIP-treated). Error bars represent means ± s.e.m. B. Sterile injury to the cornea did not induce Phase II after intraocular injection of TSG-6 (2 µg), an inhibitor of TLR2/NF-κB signalling in resident macrophages. n = 5 per group at each time point. p values, versus control (PBS-treated). Error bars represent means ± s.e.m. C. Intraocular injection of TSG-6 (2 µg) significantly suppressed the HSPB4-induced neutrophil infiltration in the cornea at 24 h after injury, suggesting the importance of TLR2/NF-κB signalling in the action of HSPB4. n = 5 per group. Error bars represent means + s.e.m. D. Also, HSPB4 induced significantly lower infiltration of neutrophils in the cornea of TLR2 knockout mice (TLR2^−/−) at 24 h after injury (n = 4 per group), which demonstrated that HSPB4 primarily acted through TLR2. Error bars represent means + s.e.m. E. Necrotic corneal extracts, but not heat-treated extracts, activated macrophages in culture to express pro-inflammatory cytokines. Treatment with polyclonal (pAb) or monoclonal (mAb) antibodies to HSPB4 significantly reduced macrophage activation by necrotic extracts, suggesting that HSPB4 in the extracts was responsible for effects on macrophage activation. n = 6 per group. p values, versus necrotic extracts-treated and antibody-untreated group. Error bars represent means + s.e.m. F. Activation of macrophages by recombinant HSPB4 was concentration-dependent. n = 6 per group. p values, versus no HSPB4 treatment. Error bars represent means + s.e.m. G. Recombinant HSPB4 caused nuclear translocation of the NF-κB complex in macrophages. H. Necrotic corneal extracts stimulated the TLR2/NF-κB pathway in cells expressing TLR2 (HEK-TLR2). Antibodies to HSPB4 significantly inhibited the effects. n = 6 per group. Error bars represent means + s.e.m. I,J. HSPB4 concentration-dependently stimulated NF-κB signalling in cells expressing TLR2, but had no effect in cells without either receptor (HEK-null). n = 6 per group. p values, versus no HSPB4 treatment. Error bars represent means + s.e.m.

Figure 6. TSG-6 inhibition of HSPB4-activated macrophages and the requirement for CD44

A,B. Dose-dependent suppression by TSG-6 of macrophages activated by HSPB4. n = 6 per group. p values, versus PBS treatment. Error bars represent means + s.e.m. C. TSG-6 had no effect on NF-κB signalling in HEK-TLR2 cells that did not express CD44. n = 8 per group. Error bars represent means + s.e.m. D.Inhibition by TSG-6 of NF-κB signalling in HEK-TLR2 cells expressing CD44. n = 8 per group. Error bars represent means + s.e.m. E.TSG-6 did not suppress the inflammatory response at 24 h after injury in transgenic mice with null alleles for CD44 (CD44^−/−), whereas TSG-6 significantly inhibited inflammation in the cornea of wild-type mice expressing CD44 (C57B/6). n = 10 per group. Error bars represent means + s.e.m.

Figure 7. Graphic summary of sterile inflammation in the cornea

Immediately after injury, SN is released from nerve endings in the cornea to recruit circulating neutrophils and induce an initial inflammatory response (Phase I). Then necrotic or injured keratocytes release HSPB4 in response to injury and oxidative stress. The HSPB4 activates resident macrophages in the cornea via TLR2/NF-κB signalling pathway to produce pro-inflammatory cytokines including IL-1 and IL-6. The pro-inflammatory signals released by resident macrophages are amplified by keratocytes that produce chemokines to further recruit large amount of neutrophils (Phase II). TSG-6 inhibits the initial activation of resident macrophages by modulating TLR2/CD44/NF-κB signalling and thereby decreases the Phase II inflammatory response.