Introduction: Imbalance in the post-traumatic inflammatory response often leads to severe complications in multiple trauma patients including increased suspectibility to sepsis post-injury. Toll-like receptors (TLRs) have been implicated in inflammatory responses after trauma. However, the role of TLR4 and TLR2 in delayed immune dysfunction following trauma such as severe skeletal injury is unknown. To study this we have developed a novel pseudofracture model which recapitulates the systemic and end organ responses observed following bilateral femur fracture and we investigated the contribution of TLR2 and TLR4 on the delayed immune dysfunction following severe trauma.

Methods: Male wild type (WT) C57BL/6, TLR2-/- and TLR4-/- mice (n=4-10), underwent pseudofracture (crushed bone solution injection and soft tissue injury to the thigh musculature bilaterally). Control mice received no experimental manipulation. At 48hrs, spleens were harvested to assess splenocyte proliferation and Th1cytokine release (IFN?, IL-2) in response to concanavalin A (2.5 µg/ml). Splenocyte proliferation was assessed using tritiated thymidine uptake as counts per minute (c.p.m.). Statistical significance (p<0.05) was assessed by Student’s t-test and ANOVA.

Results: WT mice showed a significant decrease in splenocyte proliferation at 48hrs after pseudofracture (21,258 ±1,723c.p.m.) in comparison to controls (48,102 ± 4,808c.p.m.). Splenocyte proliferation in TLR2-/- mice was similar to WT mice at baseline (46,486 ±3,371c.p.m.). However, in contrast to WT mice, splenocytes from TLR2-/- mice proliferated at the same rate as uninjured mice even after injury (40,249 ±1,900c.p.m.). We found significantly impaired splenocyte proliferation in all TLR4-/- mice, both uninjured controls (22,161 ±3,906c.p.m) and at 48hrs after pseudofracture (27,386 ±2,650c.p.m).

Splenocyte release of Th1 cytokines was also significantly decreased in injured WT mice (IL-2: 94.9 ±8.5pg/mL; IFN?: 158.0 ±31.8pg/ml) in comparison with controls as expected (IL-2: 250.3 ±18.5pg/mL; IFN?: 622.0 ±50.8pg/ml). This significant suppression was also found in the splenocytes from the injured TLR2-/- mice (IL-2: 159.6 ±5.9pg/mL; IFN?: 199.7 ±31.9 pg/ml) compared with TLR2-/- control mice (IL-2: 309.4 ±10.0 pg/mL: IFN?: 477.1 ±82.3pg/ml). However, all the TLR4-/- mice showed reduced IL-2 release, in a similar pattern that mirrored the proliferation findings (uninjured: 91.0 ±8.0pg/mL; injured: 126.8 ±6.9pg/mL). A reduced IFN? release was noted in splenocytes from control TLR4-/- mice (174.6 ±38.9pg/mL), and this was not seen in injured TLR4-/- mice where IFN? release was similarly raised to baseline WT levels (531.3 ±40.9pg/mL).

Conclusion: Our novel pseudofracture model leads to delayed immune dysfunction typical of severe injury models. Our observation that TLR2 and TLR4 deficient animals fail to develop specific aspects of the delayed immune dysfunction indicates that signaling through these TLRs contributes to the immunosuppression seen after severe trauma and represent potential as therapeutic targets to limit injury-induced immune suppression and susceptibility to sepsis post-injury.