In this study we demonstrate that a MTB virulence factor, ManLAM, is capable of inhibiting T cell migration to the lymph tissue egress signal S1P. This was accomplished by interference with PI3K/AKT pathway activation. Th1 populations were preferentially affected due to their dependence on AKT activation for S1P induced migration. It is possible that the effect of ManLAM on T cells could delay and potentially reduce initiation of Th1 effector responses in lung tissue, where even transient delays in initiation of adaptive immunity could contribute to long-term impairment for control of infection (8
). Further, these findings demonstrate a potential mechanism by which MTB could foster a favorable infection environment, and suggest that therapeutics that target ManLAM, such as benzothiazinones, may significantly alter Th1 cell skewing and recruitment to the lung thereby reducing the bacterial burden (37
We have identified that naïve and Th1 cell S1P-induced migration requires the AKT pathway, and that ManLAM pretreatment is able to inhibit S1P-directed migration of these populations. The selective effect of ManLAM on Th1 cells seems to rely on their propensity to use the AKT pathway as opposed to the ERK pathway: we found that Th1 cells have basal levels of phospho-AKT that are absent in Th2 populations, and conversely Th2 cells have basal phospho-ERK that is absent in Th1 populations. This data corroborates previous studies that have shown distinct signaling events in human Th1 and Th2 cells (38
), and that AKT and ERK contribute to Th1 and Th2 differentiation, respectively (39
). Additional studies are required to examine in more detail the mechanisms by which ManLAM desensitizes the AKT pathway.
A number of chemoattractants have been reported to induce desensitization of cells to subsequent migratory stimuli. Examples of this would be cross-desensitization of CCR5 by IL-16 and MIP-1β (41
), CD4-iduced desensitization of CXCR3 and CCR5 following IL-16 stimulation (42
), and desensitization of CXCR2 and C5aR by fMLP stimulation (43
). ManLAM has previously been shown to induce T cell migration (45
) and we now demonstrate that pretreatment with ManLAM renders T cells refractory to S1P stimulation. Most of the chemokine receptor cross-desensitization studies involved receptor mediated desensitization, and ManLAM has been shown to interact with some cell surface receptors including C-type lectins and the mannose receptor (13
). Future studies could determine whether or not ManLAM interacts with a receptor on the surface of T cells that could mediate this effect. Another potential mechanism is the direct incorporation of ManLAM into the membranes of PBMCs: Ilangumaran et. al. showed that ManLAM preferentially incorporates into lipid rafts where AKT activation following S1P1
stimulation normally occurs (16
). Our data suggest that ManLAM can selectively inhibit the AKT pathway without abrogating ERK signaling. ERK signaling has been shown to be caveolae independent and therefore potentially less affected by membrane integration by ManLAM (46
). This difference may explain why ManLAM preferentially inhibits the PI3K pathway; however, additional studies are required to determine the mechanism by which ManLAM binds to T cells and initiates the inhibitory effect on S1P receptor signaling.
Our studies did not address effects of ManLAM on other S1P receptors expressed by other immune cells. Of note, there are 5 different S1P receptors that are differentially expressed on immune cells (47
). Our studies focused on T lymphocytes (48
), which express S1P1
as do B lymphocytes; however, mast cells, macrophages and dendritic cells express S1P2
and ManLAM stimulation either through membrane integration or receptor interaction could affect cellular responses. Further examination of this phenotype could reveal preferential use of different S1P receptors on different leukocytes (49
). It has also been shown that MTB inhibits sphingosine kinase activity, the enzyme responsible for production of S1P (50
). It is possible that reduced production of S1P in vivo
, a consequence of MTB infection, could contribute to the phenotype we observe.
Previous studies have shown that 100ng/mL of ManLAM represents the lower end of a dose response curve used to inhibit CD4+
T cell activation and cytokine secretion (20
). In addition, Human MTB patient sputum has been shown to contain 1 ng/mL to 1 μg/mL of ManLAM (30
), though the precise concentration in lymph nodes is unknown. It has been reported that ManLAM incorporates into lipid rafts on the surface of PBMCs as rapidly as 30 min, with maximal incorporation occurring within several hours (16
). Concentrations and timepoints used in this study correspond well with these studies, supporting the concept that ManLAM can alter the immune response in the nanogram range and within a short period of time. During the course of a live infection, it is likely that ManLAM is constantly secreted by infected macrophages, thus potentiating the sequestration of cells for an extended period of time. The spread of infection to the lymph nodes and subsequent ManLAM secretion within the confines of the lymph tissue could further contribute to inhibition of Th1 cell egress with reduced immune responses in the lung. Interestingly, MTB infected CCR5−/− mice demonstrate increased lymphocytic infiltrates in their lungs and increased proinflammatory cytokine production (52
), which would be consistent with ManLAM’s ability to efficiently and selectively inhibit CCR5+/Th1 cell subsets but not CCR4+/Th2 subsets.
There is evidence that indicates a shift in Th1/Th2 cytokines during MTB infection. Infected mice initially produce both IFNγ and IL-4 in the lungs, but IFNγ levels decrease while IL-4 levels increase over the course of infection (53
). This finding is consistent with our data indicating that ManLAM production would alter normal Th1 cell homing patterns. ManLAM has also been shown to promote secretion of Th2 related cytokines while simultaneously decreasing release of Th1 cytokines from T lymphocytes (34
). As IFNγ production by Th1 cells is considered to be host-protective, our data would support the hypothesis that shedding of ManLAM during MTB infection promotes an environment that is more favorable for infection, and suggests further that antibiotics targeting ManLAM, such as benzothiazinones, may significantly alter Th1 cell skewing in the lung thereby reducing the bacterial burden (37
). It is likely that live MTB produce other factors that are immunoregulatory. Our data indicate that shedding of ManLAM represents a major component for induction of lymphadenopathy with selectivity for CCR5+ T cells; however, additional studies are required to more fully understand how bacterial products can modify the immune response and promote the infection process.