Our results show for the first time that the adipocytokine leptin plays a protective role in mucosal resistance to amebic infection, and does so primarily via leptin receptors expressed on intestinal epithelial cells. Furthermore, both the Y1138/STAT3 and Y985/SHP2/ERK signal transduction pathways of LRb are required for leptin-mediated protection.
The role of leptin in promoting host resistance to infection has been demonstrated in several studies using leptin- and leptin receptor-deficient mice 38–40
. Prior studies implicated the pro-inflammatory effect of leptin on cell-mediated immunity as being important for control of infection. However, leptin is a pleiotropic hormone that regulates the neuroendocrine axis centrally and immune/epithelial homeostasis peripherally 3, 7, 8
. While the pro-inflammatory cellular response may contribute to the clearance of the infection in some circumstances, the effects of leptin on prevention of epithelial apoptosis and promoting tissue repair might be required for mucosal defense against pathogens like E. histolytica
. In our study, leptin appeared to be an essential factor for host resistance to intestinal amebiasis, as supported by the enhanced susceptibility in ob/ob and db/db mice. Compared to the highly resistant wild type B6 controls, an increase in mortality rate was observed in db/db and ob/ob mice after E. histolytica
infection, and the mice exhibited profound mucosal destruction in the intestine. Interestingly, although no statistical significance was reached, the receptor deficient db/db mice appeared to be more susceptible to both infection and mortality compared to the leptin deficient ob/ob mice. This finding suggested that the leptin receptor may signal to some extent in the absence of leptin, or the existence of additional ligands other than leptin that might partially compensate the leptin deficiency in ob/ob mice. A similar pattern has been observed for Ciliary Neurotropic Factor (CNTF), where mice lacking the CNTF receptor, rather than mice lacking CNTF, exhibited profound motor neuron deficits at birth 41
. Our data, together with the previous observations that ob/ob mice have enhanced sensitivity to endotoxin-induced lethality 42
and organ failure 43, 44
, suggest that leptin may be involved in the acute phase response to systemic inflammation and mucosal injury.
The connection between leptin deficiency and disease susceptibility is likely to be complex. The functional leptin receptor (LRb or OB-Rb) is expressed on many immune cell types. Leptin has been shown, for instance, to modulate T cell proliferation, promote Th1 responses, and protect a variety of cell types from apoptosis 3, 8, 11–13, 45
. These observations led to the speculation that lack of effects of leptin on immune cells might account for the impaired resistance to amebic infection observed in ob/ob and db/db mice. In order to determine the relative contribution of leptin effects on the immune versus non-immune system, we examined the susceptibility of bone marrow chimeras (BMCs) between db/db and wild-type B6 mice. Both the infection and mortality rates were similar in db/db mice reconstituted with either db/db or wildtype bone marrow, indicating that leptin actions on radiosensitive hematopoietic cells are not sufficient to confer protection. Accordingly, LRb expressed on radioresistant cells, such as neurons or peripheral non-immune cells, may play an essential role in protection.
LRb-expressing neurons in the brain have the potential to regulate susceptibility to infection directly or indirectly. With regard to the former, leptin could act centrally by regulating the activity of nerves projecting from the CNS to immune organs or the intestine for example. Alternatively, leptin’s actions could be an indirect secondary effect of the multiple neuroendocrine and metabolic abnormalities that result from lack of central LRb function. For example, lack of central leptin function in ob/ob and db/db mice increases activation of the hypothalamic-pituitary-adrenal axis, leading to increased glucocorticoid and decreased thyroid and growth hormone levels which could indirectly affect immune and intestinal functions important in amebic infection 46
. To address the contributions of central leptin signaling to leptin-mediated protection, Tam-Cre mice with an intact leptin receptor present in the brain but disrupted in periphery were generated and tested for their susceptibility to amebiasis. An increase in both susceptibility and disease severity were observed in Tam-Cre mice compared with non-cre littermates, indicating that intact central leptin signaling alone is not sufficient to protect the host from mucosal infection, further pointing to the importance of intact LRb signaling in the periphery for protection. LRb expression has been detected in the gut epithelium 25, 47
, together with the fact that mucosal destruction in db/db mice happens within 24hrs, suggesting that leptin might be acting locally on intestinal epithelial cells to mediate protection. This hypothesis was finally verified by infecting mice harboring a targeted deletion of lepr
in intestinal epithelial cells (Vil-Cre KO), and showing that they exhibited significantly higher susceptibility and a greater level of mucosal destruction compared to their littermate controls. Taken together, these data demonstrate that leptin-mediated protection from amebiasis is mediated primarily through a direct action on the epithelial cells of the intestinal mucosa.
Some insight into the mechanism by which leptin mediates mucosal protection has been gained from our genetic studies that identified Q223R, a common genetic polymorphism in the leptin receptor gene, to affect the susceptibility to amebiasis (Duggal, Guo & Petri, unpublished). Children with the R allele showed significantly higher susceptibility than subjects homozygous for the Q allele, and this association was further verified in transgenic mice showing that the R allele carriers were more susceptible to amebic infection along with a greater level of epithelial apoptosis and reduced Akt phosphorylation in the gut. The leptin receptor has anti-apoptotic effects via its activation of the PI3K/Akt pathways 17, 28, 29
, and E. histolytica
is known to kill host cells by inducing caspase 3 activation 48, 49
. We therefore hypothesize that the ancestral leptin receptor with the favorable Q allele protects the intestinal epithelium from E. histolytica
induced apoptosis by activating the PI3K/Akt pathway.
Increased susceptibility to amebiasis was also observed in both S1138 and L985 variants. The S1138 variant has impaired LRb-STAT3 signaling and disrupted expression of a variety of STAT3-inducible genes; the L985 mutant on the other hand, has enhanced STAT3 activation due to the loss of inhibitory signaling. It has been shown that the hypothalamic signaling of leptin through STAT3 plays a critical role in regulation of neuroendocrine functions 31, 50
, which explains the obesity and infertility phenotype of the S1138 mouse. However, how the LRb-STAT3 signaling might function in periphery remains unclear. Previous studies have shown that the IL-6/gp130/STAT3 signaling axis is important in maintaining intestinal homeostasis as 1) Mucosal inflammatory cells responding to IL-6/IL-6R trans-signaling contribute to the development of colitis in a STAT3-dependent pathway 51, 52
; and 2) Enterocyte-specific STAT3 activation promotes epithelial survival and proliferation 53, 54
. As an IL-6 receptor family member, leptin receptor is presumably acting on the intestinal mucosa in a similar pattern. The enhanced susceptibility of S1138 mice to intestinal amebiasis indicated the importance of LRb-STAT3 signaling in leptin-mediated mucosal defense, but the downstream events accounting for the protection remain unknown. Studies are underway to identify the candidate effectors that are inducible by LRb-STAT3 and involved in the local mucosal defense (i.e. anti-apoptosis, pro-proliferation and anti-microbial peptide production).
The blocked inhibitory signaling in the L985 variant has been reported to result in enhanced STAT3 activation and accordingly a “gain-of-function” leptin-hypersensitivity phenotype characterized by low body weight, low circulating leptin, and resistance to diet-induced obesity 32, 33
. It has been demonstrated that the strength and duration of STAT3 activity determines if it is anti- or pro-apoptotic, and enhanced STAT3 signaling due to loss of SOCS3 could induce apoptosis via repressing the PI3K/Akt pathway and reducing Bcl-2 and Cyclin D1 expression 55
. This finding could partly explain why we saw an increased susceptibility to amebiasis in the L985 mice with disrupted SOCS3 binding and enhanced STAT3 signaling. Another important pathway through LRb Y985 is SHP2-GRB-MAPK/ERK, whose functional effect in vivo has not been well defined. The uncoupled SHP2 binding to the L985 mutant disrupts MAPK/ERK activation, which has been considered as another pro-survival signal (in addition to PI3K/Akt) and is implicated in the survival of various types of epithelial cells 56–58
. Thus the increased susceptibility in L985 mice may be via attenuated MAPK/ERK signaling due to the L985 mutation. Note that the cecal histopathology was different for the L985 and S1138 infected mice (), suggesting the divergent effects of leptin action in control of epithelial resistance.
Taken together, it is possible that multiple leptin signaling pathways are involved in protection against intestinal amebiasis, and the mechanisms by which 223R LRb fails to protect might not be the same for the S1138 and L985 receptors. Further study is necessary to clarify the relationship between STAT3/SOCS3, MAPK/ERK and PI3K/Akt pathways in leptin-mediated resistance to amebiasis.
In conclusion, the data presented here indicate that differential susceptibility to amebiasis can be explained at least in part by variable leptin receptor function; Leptin-mediated resistance is via its actions on intestinal epithelium rather than hematopoietic cells or the brain, and receptor signaling through STAT3 or SHP2/ERK are both involved in leptin-mediated resistance to E. histolytica infection. By showing the association of the adipocytokine leptin with susceptibility to a mucosal parasitic infection, and by demonstrating its site of action and signaling pathways, our work provides insights into the mechanisms by which malnutrition compromises the immune system.