In this report, we have observed that oral laquinimod treatment can prevent or reverse established EAE. In vivo laquinimod treatment was associated with alterations in myeloid APC subpopulations that included a reduction in CD4+
cDC, a potent DC subpopulation. We have shown that laquinimod treatment promoted development of anti-inflammatory type II monocytes and DC, reminiscent of our previous work demonstrating that in vivo treatment of mice with glatiramer acetate (GA, copolymer-1, Copaxone®), an approved therapy for RRMS 
, induced differentiation of anti-inflammatory type II monocytes 
. Laquinimod did not alter numbers of CD4+
T cells. In a previous study, it was observed that laquinimod treatment reduced secretion of IFN-γ and IL-17 
. Here, we have demonstrated that oral laquinimod administration in EAE was associated with anti-inflammatory T cell polarization as demonstrated by reductions in frequencies of proinflammatory Th1 and Th17 cells in vivo, and an increase in Treg. By studying how in vivo laquinimod treatment influenced individual populations of myeloid APC or naive myelin-specific T cells, we demonstrated that T cell immune modulation was linked to induction of type II myeloid APC, but not from its effects on T cells alone. Thus, at physiologic levels achieved by in vivo treatment, laquinimod impacts APC, but may not influence T cells directly.
In general, CD4+
T cells, which express antigen-specific α/β+
T cell receptors, recognize peptide fragments of processed proteins only in association with polymorphic MHC molecules on APC 
. In this regard, GA, which is a polypeptide-based therapy, provides antigenic determinants and leads to expansion of GA-reactive T cells that can be identified in therapy of MS 
or EAE 
. The requirement for MHC II on GA-induced type II monocytes was discovered at the time adoptive transfer of monocytes was first developed as an experimental paradigm to study how therapeutics can influence APC-T cell interaction in vivo 
. In this regard, GA-induced type II monocytes from wild-type mice, but not from mice selectively deficient in MHC II, induced T cell immune modulation (i.e. expansion of Treg and Th2 cells) and reversed EAE in recipient mice. However, as a synthetic heterocyclic molecule, laquinimod itself is unlikely to serve as a target for T cell recognition. Use of the EAE model permits investigators to characterize how laquinimod can alter T cell responses that are elicited by direct immunization with myelin peptides or proteins, a situation not encountered in MS, a naturally occurring disease. Evaluating whether laquinimod treatment of MS modulates T cell function in the absence of active antigenic stimulation may be more challenging. Our observations that in vivo laquinimod treatment of unimmunized mice modified expression of myeloid subpopulations and APC function should focus attention on exploring the mechanism of action of laquinimod in MS therapy on cells of the innate immune system. Our demonstration that laquinimod has a principle effect on innate immunity provides mechanistic insight relevant to results from the two recent phase III clinical trials in RRMS that tested laquinimod (0.6 mg daily) and indicated that dose provided a more pronounced effect on disability progression than relapse rate reduction 
. Further, our findings also suggest that laquinimod could be beneficial in secondary progressive MS, a phase that involves chronic inflammation and neurodegeneration that is thought to be driven by innate immunity 
A recent study evaluated the potential role of brain-derived neurotrophic factor (BDNF) in laquinimod treatment 
. A small, but significant increase in serum BDNF levels was detected in laquinimod-treated MS patients. These authors also evaluated the role of BDNF in laquinimod treatment of EAE. It is known that BDNF-deficient mice develop more severe chronic EAE 
. They demonstrated that monocytes from laquinimod-treated donor wild-type mice, but not monocytes from laquinimod-treated BDNF-deficient mice or from untreated wild-type mice, ameliorated EAE in recipient mice. The authors concluded that the effects of laquinimod on monocytes are BDNF-dependent. However, they did not transfer untreated BDNF-deficient monocytes, and therefore did not distinguish the influence of the production, or the absence, of BDNF alone on monocyte function independent of laquinimod treatment. In order to attribute the effect of laquinimod to BDNF production by monocytes using this experimental approach, it is advantageous to not only compare laquinimod-treated and untreated wild-type monocytes to laquinimod-treated BDNF-deficient monocytes, but to simultaneously compare the adoptive transfer of untreated BDNF-deficient monocytes to both untreated wild-type monocytes and to laquinimod-treated BDNF-deficient monocytes.
In this investigation, we have identified cellular mechanisms that contribute to immune modulation by laquinimod, focusing on the interaction of myeloid APC and T cells. Type II monocyte differentiation was associated with reduced production of proinflammatory IL-6, IL-12/IL-23 (p40) and TNF, and increased production of anti-inflammatory IL-10. It is important to characterize the molecular pathway(s) utilized by laquinimod for this type II cytokine profile. Laquinimod is not known to have a well-defined target, although some in vitro data suggest that quinoline-3-carboxamides bind S100A9, a calcium binding protein 
that influences cell signaling. Other results indicate that this class of molecules may alter NF-κB signaling 
. We have begun evaluating the signaling events contributing to type II APC differentiation in monocytes/macrophages isolated from laquinimod-treated mice. First, we focused on activation of STAT1, a transcription factor that participates in expression of several proinflammatory cytokines 
. Laquinimod treatment suppressed inducible STAT1, but did not alter activation of p38 MAPK, another signaling pathway involved in expression of proinflammatory cytokines that can be regulated independently or coordinately with STAT1 
. Interestingly, inhibition of STAT1 and p38 MAPK signaling was observed in development of type II monocytes by GA 
(N. Molnarfi and S.S. Zamvil, unpublished), suggesting that the signaling events modulated by GA and laquinimod during type II APC differentiation are not the same. Our observations represent only the initial steps in understanding how laquinimod influences intracellular signaling pathways in type II myeloid cell differentiation. Although we used both in vivo and ex vivo analyses to evaluate type II myeloid cells, laquinimod was only administered in vivo, which we believe more closely reflects the physiology of laquinimod treatment in MS. In contrast with previous studies 
, we evaluated APC-T cell interaction, the interface between innate and adaptive immunity, primarily by in vivo laquinimod treatment in the absence of peptide immunization, and therefore obviating concern of adjuvant. Our findings in this report support evaluation of type II myeloid cells in laquinimod treatment of MS patients.