Macrophage effector functions controlled by Bruton's tyrosine kinase are more crucial than the cytokine balance of T cell responses for microfilarial clearance

Abstract

Macrophages from X-linked immunodeficient (xid) mice lacking functional Bruton’s tyrosine kinase (Btk) show poor NO induction and enhanced IL-12 induction and contribute to delayed clearance of injected microfilaria (mf) in vivo. We now show that Btk is involved in other macrophage effector functions, such as bactericidal activity and secretion of proinflammatory cytokines (TNF-α, IL-1β), but not the T cell-directed cytokine IL-12. Induction of some transcriptional regulators of the NF-κB family, crucial for the expression of proinflammatory cytokines, is also poor in Btk-deficient macrophages. Thus, Btk appears to be involved in signaling for inducible effector functions, but not APC functions, in macrophages. Furthermore, adoptive transfer of T cells from mf-infected xid or wild-type mice did not alter the course of mf clearance in recipients, mf clearance was unaltered in IFN-γ-deficient mice and improved mf clearance was seen only if greater inducibility of IL-12 was accompanied by greater NO secretion from macrophages, as seen in Ityr C.D2 mice as compared with congenic Itys BALB/c mice. Thus, delayed mf clearance in xid mice was correlated not with the high IL-12/Th1 phenotype but with low NO induction levels. Also, xid macrophages showed poor toxicity to mf in vitro as compared with wild-type macrophages. Inhibition of NO production blocked this mf cytotoxicity and an NF-κB inhibitor blocked both NO induction and mf cytotoxicity. Thus, Btk is involved in inducing many macrophage effector functions and delayed mf clearance seen in Btk-deficient xid mice is due to poor NO induction in macrophages, resulting in compromised microfilarial toxicity. The role and nature of immune-mediated protection in metazoan infections such as filariasis are still unclear (1, 2, 3, 4, 5, 6). The role of the Th1/Th2 cytokine balance in the T cell response and the relative role of the innate and adaptive immune responses are major issues of debate (7, 8, 9, 10, 11, 12, 13, 14), particularly because T cell cytokines (15, 16, 17) as well as certain innate immune mechanisms (14, 18, 19, 20) have been suggested to be trophic for filarial development as opposed to protection. Animal model-based studies of antifilarial immunity have been compromised by the fact that fully susceptible animal models such as gerbils, jirds, multimammate rats or larger animal species (21) are not yet easily amenable to detailed immunological analyses. Immunological studies performed in immunocompromised mice have been very useful in dissecting the nature of immunity in experimental filariasis, notwithstanding the fact that even such mice are not always fully susceptible to development of filarial parasites (21, 22). However, in immunocompetent mice, i.p. injected microfilaria (mf)4 can survive in peripheral blood for about 4 wk (23), although in T cell-deficient nude or SCID mice they survive for much longer times, establishing that active immunity is responsible for accelerated clearance, although the role of various components of the immune system in such clearance is still unclear. We have reported previously that X-linked immunodeficient (xid) mice lacking functional Bruton’s tyrosine kinase (Btk) show prolonged persistence of circulating mf (16). Anti-mf T cell proliferative responses are enhanced and the T cell cytokine pattern is skewed in the Th1 direction with relative dominance of IFN-γ in xid mice in comparison to wild-type mice (16). This bias is due to decreased induction of inducible NO synthase (iNOS) in the xid macrophages due to the absence of functional Btk, leading to derepression of IL-12 induction, in turn causing the observed Th1 cell cytokine bias (24). What is the role of the observed Th1 bias in anti-mf T cell responses found in xid mice in their inability to clear mf rapidly? There is some controversy about the precise role of the T cell cytokine balance in filarial infection. The T cell responses of asymptomatic humans with microfilaraemia show a prominence of Th2 cytokines such as IL-4, while T cells from symptomatic patients of elephantiasis show a relative dominance of the Th1 cytokine IFN-γ, as do some putatively immune “endemic normal” individuals (2, 12). However, Th2 responses appear to be protective in some experimental animal models (10, 25, 26, 27). In contrast, our previous data also show that some macrophage effector functions may be compromised in Btk-deficient macrophages, because NO induction is severely inhibited (24). The mf are highly susceptible to NO and peroxynitrite-mediated damage in vitro (28) and mf clearance in mice can be delayed by administration of aminoguanidine (AG) (29), an inhibitor of iNOS (30, 31). However, we have also observed that NO negatively regulates IL-12 induction (24) and it therefore remains possible that the role played by macrophage NO in the mf clearance defect in xid mice is connected to Th1 cytokine biases rather than to macrophage effector functions. On this background, we have investigated the effect of Btk deficiency on macrophage effector functions as well as the roles played by Th1 anti-mf T cell responses, particularly with reference to the role of IFN-γ, in mf clearance. We show in this study that the induction of macrophage effector functions is selectively inhibited by Btk deficiency and, while mf clearance rates remain unaffected by Th1 cytokine patterns or the lack of IFN-γ, they are critically dependent on macrophage effector functions, particularly on NO induction.