The present study demonstrates that LITAF plays an important role in the regulation of TNF-α secretion in the inflamed colon. Following a TNBS inflammatory stimulus, LPM harvested from acutely inflamed colonic tissue secreted higher basal levels of TNF-α compared to LPM harvested from non-inflamed tissue; LPS was found to further increase this TNF-α secretion. These findings are further strengthened by our data demonstrating that TNF-α secretion is significantly reduced in LPM harvested from the inflamed colons of LITAF mac -/- knockout mice in which the LITAF gene has been selectively deleted from macrophages. The present data constitute the first demonstration that LITAF mRNA expression and protein levels are increased in LPM isolated from inflamed colonic tissues compared to non-inflamed colonic tissues, strongly supporting the likelihood that LITAF mediates in part the increase in TNF-α secretion. Collectively the present data implicate LITAF in the regulation of TNF-α secretion.
In order to compare macrophage TNF-α secretion responses following colonic inflammation with and without LITAF, it was necessary to use C57BL/6 mice because LITAF mac -/- mice were generated in same strain.
Although C57BL/6 mice are generally resistant to TNBS-induced inflammation
it has been shown that this mouse strain does exhibit a quantifiable inflammation during the first 24–48 hours following TNBS administration.
This feature of milder degree of inflammation permitted the isolation of viable LPM for analyses ex vivo
. LPM harvested from inflamed colonic tissue have advantages over monocyte/macrophage cell lines, such as THP-1 cells, or macrophages specific to other organs since the specific phenotype is dictated by the microenvironment in which a macrophage matures.
Primary isolation of LMP also allows for the in vivo
activation of the specific cells which under non-inflamed conditions show a hyporesponsiveness to LPS.
Given the short duration of the colon inflammation studied (24 hours), it is highly probable that the LPM harvested are resident LPM and are not newly recruited monocytes and macrophages whose recruitment to inflamed tissue has been determined to occur from 2 to 10 days following injury.
Inflammatory signaling in macrophages begins with activation of the Toll like Receptor 4 (TLR4) which leads to activation of Mitogen Activated Protein Kinase (MAPK) pathways, including p38.
Downstream targets of p38 include numerous transcriptional regulators, including AP-1 and C/EBP-1.
In addition, p38 has been shown to activate LITAF with no significant changes of TNF-α protein levels in macLITAF−/−
cells either treated with LPS alone or cotreated with SB203580 plus LPS.
Inhibitors of p38 were used in this ex vivo
system to determine if inhibition could reduce TNF-α secretion from LPM harvested from inflamed colonic tissue. A commonly used p38 specific inhibitor SB-202190 
partially reduces the TNF-α response in LPM harvested from inflamed colonic tissue following LPS stimulation. While p38 inhibition likely leads to many downstream effects, the observed reduction in TNF-α secretion is consistent with an inhibition of the LITAF contribution to TNF-α secretion.
Additionally, TLR4 is known to activate the NF-κB pathway through the ubiquitination and degradation of IκB. IκB degradation allows for the translocation of the p65 and p50 heterodimeric unit to the nucleus where transcription of inflammatory genes, including TNF-α, is induced.
In this ex vivo
system, inhibitors of NF-κB signaling also caused a reduction in TNF-α secretion in response to LPS stimulation in LPM harvested from inflamed colonic tissue. This is not surprising given that NF-κB is a ubiquitous transcription factor that regulates over 300 genes, including TNF-α, many involved in inflammation and cancer.
The human TNF-α promoter region contains multiple NF-κB consensus sequences, in addition to consensus sequences for many other transcription factors, including SP-1 and Erg1,
AP-1 and AP-2,
Although not definitive, taken together, our data lend support to our hypothesis that the increased TNF-α response in LPM harvested from inflamed colonic tissue may stem from multiple pathways including NF-κB, LITAF and p38 MAPK pathways.
The most direct evidence that LITAF is associated with the capacity of LPM to secrete TNF-α comes from the use of LITAF deficient LPM in these studies. Using LPM harvested from LITAF mac -/- mice following TNBS induced inflammation, the data supports the conclusion that the deletion of the LITAF gene leads to a significant reduction in TNF-α secretion in response to LPS stimulation compared to wildtype controls. Since differences in TLR4 mRNA levels were not found between peritoneal macrophages derived from C57BL/6 and LITAF mac -/- mice (data not shown) it very likely that this process is not dependent on changes in TLR4. In this system, the remaining TNF-α signal that we have observed in LITAF deficient macrophages is likely due to the remaining proinflammatory pathways including NF-κB.
LITAF mac -/- mice exhibit reduced bodyweight loss, have lower MPO levels and, on examination of their colonic tissue, express less TNF-α mRNA as compared to C57BL/6 mice. Despite the apparent reduction in the acute TNBS-induced inflammatory response, LITAF mac-/- mice do not exhibit a reduction in the overall histological damage to the colonic tissue following 24 or 48 hours after TNBS administration. These findings suggest that the animals deficient in LITAF have a delayed healing response compared to C57BL/6 mice. This may be due, in part, to the rapid and strong inflammatory stimulus that TNBS administration imparts when administered rectally. Another explanation for this result may be related to a beneficial dysregulation of the inflammatory response in LITAF mac -/- animals.
Indeed LITAF mac -/- mice have been shown to have reduced levels of many inflammatory cytokines and chemokines, when administered a sublethal dose of LPS. Compared to the early surge of serum inflammatory cytokines seen in wildtype mice, LITAF mac -/- mice show a delayed increase of proinflammatory cytokines, and a persistent increase of anti-inflammatory cytokines illustrating that the absence of LITAF leads to a beneficial temporal dysregulation of the inflammatory response.
The histological inflammatory response observed 24 hours after the acute inflammatory stimulus of TNBS in LITAF mac -/- mice may just reflect a delay of inflammation consistent with the previous report 
Interestingly, while making synthetic derivatives of the lactone kavain, Pollastri et al.
identified an acyclic derivative ((E
)-5-biphenyl-4-yl-5-hydroxy-3-methoxy-pent-2-enoic acid methyl ester) and showed this compound to suppress LITAF levels while simultaneously reducing TNF-α secretion from macrophages stimulated with LPS.
Future studies may lead to optimization of this compound and may increase our understanding of the feasibility of the use of kava compounds to specifically inhibit LITAF and further explore anti-LITAF strategies as a potential treatment for inflammatory diseases known to be associated with increased TNF-α.
In summary, endogenous colonic LPMs increased their base line and LPS mediated TNF-α secretion by a LITAF dependant pathway when harvested from acutely inflamed colonic tissue suggesting the usefulness of anti-LITAF drug development for the treatment ulcerative colitis. We propose that LITAF plays a substantial role in TNF-α secretion from LMP isolated from an acutely inflamed colon and represents an alternative pathway to modulation of the TNF-α component of the inflammatory response. A better understanding of the role that LITAF signaling plays in regulating TNF-α gene expression in the LPM of the inflamed colon may provide alternative targets for therapeutic interventions not only in IBD, but other chronic inflammatory diseases exacerbated by elevated TNF-α levels.