Enteric nematode infection is associated with CD4+ T cell-dependent infiltration of a number of immune cells including eosinophils, mast cells, and macrophages at the area of infection. In the present study, we demonstrate that this immune-mediated recruitment and development of AAMϕ in the small intestine is dependent largely on IL-4/IL-13 and activation of Stat6. More importantly, these AAMϕ link Th2 cytokine production to infection-induced alterations in gut function. A distinguishing feature of AAMϕ is the metabolism of L-arginine to ornithine via arginase I. This study is the first to show that AAMϕ and the arginase pathway play a significant role in gastrointestinal smooth muscle responses to nematode infection.
Intestinal macrophages are continuously replenished by circulating monocytes1
and play key roles in mucosal homeostasis as well as host defense. Monocytes entering the healthy intestinal mucosa acquire a specific “tolerogenic” phenotype characterized by a down regulation of receptors for LPS, and the toll like receptors as well as specific chemokine receptors17, 18
. In response to enteric pathogens, cytokine-induced up-regulation of MCP-1 plays a key role in recruitment of additional circulating monocytes to the intestine and differentiation of these infiltrating macrophages. Their location in the lamina propria is strategic in the mucosal response to pathogens that cross the epithelial barrier. There is also a population of resident macrophages in the smooth muscle layer that were shown to impact smooth muscle function in response to endotoxin/LPS19
or oxidative stress20
. It is well established that macrophages undergo alternative activation in the context of strong Th2 cytokine environments, including helminth infection, asthma, or allergy2
. Consistent with previous studies, we showed here that N. brasiliensis
infection induced an increased infiltration and accumulation of AAMϕ, characterized by up-regulation of F4/80, CD206, arginase I, FIZZ1, and YM1. The requirement of Th2 cytokines IL-4- or IL-13-mediated activation of Stat6 for infection-induced accumulation and activation of AAMϕ was established by showing that N. brasiliensis
failed to up-regulate AAMϕ markers in Stat6−/−
mice. The significantly elevated AAMϕ marker mRNA expression in IL-4−/−
mice indicates that either cytokine alone is capable of eliciting the full development of AAMϕ.
Intestinal macrophages are involved in both innate and adaptive immune response. A recent study implicated AAMϕ in the innate immune response to N. brasiliensis
infection in the lung showing that early up-regulation of marker expression was dependent on Th2 cytokines released from resident granulocytes rather than T cells21
. We used SCID mice to investigate the role of the innate versus adaptive immune response to infection-induced recruitment and development of AAMϕ in the small intestine. N. brasiliensis
-infected SCID mice showed a small, but significant, increase in IL-4 and IL-13 expression as well as an increased infiltration of macrophages. These macrophages, however, did not express markers of the AAMϕ phenotype suggesting that the levels of IL-4 and IL-13 in SCID mice are insufficient to induce development of AAMϕ. It is of interest, that treatment of SCID mice with exogenous IL-13 was able to induce the AAMϕ phenotype and increase smooth muscle hyper-contractility. Unlike N. brasiliensis
infection, IL-13 did not appear to increase macrophage infiltration. Thus, IL-13 has the ability to work independently of the adaptive immune system to induce an increase in smooth muscle function that is associated with an elevated expression of AAMϕ markers in resident macrophages.
The results of the current study are consistent with other reports showing that AAMϕ influence the immune outcome during infections. Alternatively activated macrophages are linked to the suppression of T cell responsiveness in chronic infections22
and are important for down regulation of Th2 mediated immune pathology. In the present study, macrophage depletion did not change either Th1 (IFN-γ) or Th2 (IL-4, IL-13) cytokine expression, suggesting that macrophages are not involved in the initiation of the cytokine response elicited by N. brasiliensis
infection, but are part of the downstream events in response to increased Th2 cytokine production.
Host resistance to enteric parasites is associated with significant Th2- and Stat6-dependent changes in intestinal physiology8
. We and others showed previously that infection with several different enteric nematodes induced a stereotypic elevation in intestinal smooth muscle responses to acetylcholine, serotonin, agonists of protease-activated receptors, and to nerve stimulation7, 8, 14, 16, 23
. The contribution of macrophages to Th1-mediated changes in smooth muscle contractility is well established20, 24
. To determine the functional role of AAMϕ on N. brasiliensis
infection, we used Cl2
MDP-liposome to deplete macrophages. Cl2
MDP treatment effectively depleted not only infection-elicited, but also the resident macrophages evidenced by the decreased expression of macrophage markers in both infected and uninfected mice. Although macrophage-depleted uninfected mice displayed similar intestinal smooth muscle function to that untreated controls, the intestinal smooth muscle hyper-contractility observed in N. brasiliensis
-infected mice treated with PBS-liposomes was absent in Cl2
MDP-treated infected mice. These data indicate that the resident macrophages do not play a major role in the constitutive regulation of intestinal smooth muscle contractility, but are required absolutely for the hyper-contractility in nematode infection. Additionally, these macrophage-depleted mice had an impaired ability to expel worms, confirming the contribution of AAMϕ in host defense against nematode infection6
albeit by more than one mechanism. Infection also induces a STAT6-dependent increase in the smooth muscle responses to 5-HT, an effect associated with an up-regulation of the 5-HT2A
. The present study shows that this elevated 5-HT2A
expression is dependent, in part, on the presence of AAMϕ emphasizing the importance of the interaction between macrophages and smooth muscle in the Th2-mediated hyper-contractility to 5-HT.
Alternatively activated macrophages express/secrete a number of proteins that could be responsible for the protective effects of macrophages against nematode infection. Of these molecules, arginase I is the enzyme distinguishes AAMϕ from CAMϕ, leading to hydrolysis of L-arginine to ornithine25
, a precursor for polyamine biosynthesis via ornithine decarboxylase, or proline via ornithine aminotransferase. Both of these pathways are implicated in cell proliferation and collagen production. Indeed, increased arginase activity is linked to airway hyper responsiveness or decreased airway smooth muscle relaxation in asthma26, 27
. Moreover, elevated arginase I expression increases airway smooth muscle proliferation by mechanisms involving the production of polyamines28
. Although up-regulation of arginase I is a common feature in nematode infection29
, its function in host defense against nematode infection remains unclear. We showed here that administration of BEC in N. brasiliensis
-infected mice abolished the intestinal smooth muscle hyper-contractility and impaired worm expulsion. Although BEC also inhibits arginase II, the significantly down-regulated arginase II versus up-regulated arginase I expression in N. brasiliensis
-infected mice suggests that the effects of BEC were mainly through the inhibition of arginase I. The precise mechanism by which arginase regulates intestinal smooth muscle function was not investigated in the current study. Both arginase I and NOS use the same substrate L-arginine suggesting that arginase inhibition could affect the NO production. It is, however, unlikely that NO production is altered in BEC-treated mice as BEC had no effect on smooth muscle contractility in control mice. The results of the present study indicate that influx of AAMϕ with increased arginase activity play a major role in infection-induced hyper-contractility and increased smooth muscle thickness.
In conclusion, the results of the present study link the up-regulation of Th2 cytokine and activation of Stat6 with the accumulation of AAMϕ that control smooth muscle contractility and morphology via arginase I metabolism of arginine. We recently observed that mice deficient in IL-13Rα1 express arginase I after infection with N. brasiliensis
but do not expel worms from the intestine30
. This is consistent with the observation that IL-4−/−
mice express CD206, FIZZ1, and arginase I after N. brasiliensis
infection, but only IL-13−/−
mice fail to expel worms. Thus, AAMϕ, like eosinophils, mast cells, and goblet cells that develop in response to nematode infection in the intestine, respond to orchestrated cues in the local environment to affect functional activity of surrounding cells such as smooth muscle and also contribute to protective immunity against infection.