Abstract

Tumor necrosis factor (TNF)–α is required for host defenses against a number of pathogenic microbes, including Histoplasma capsulatum. In mice, TNF-α is necessary for the generation of a protective immune response to both primary and secondary histoplasmosis. Recent reports indicate that, in humans, treatment with inhibitors of TNF-α is associated with disseminated histoplasmosis. Here, I review the mechanisms by which inhibition of TNF-α may exacerbate infection with H. capsulatum.

Histoplasma capsulatum is a dimorphic fungus that is widely distributed in nature but is found principally in the Americas. Within the continental United States, H. capsulatum is endemic to the Midwest and Southeast, where it exists in the soil as a mycelium. After inhalation of spores into mammalian lungs, the fungus transforms into a pathogenic yeast form. Initial contact with the fungus usually results in either no illness or an influenza-like syndrome. One of the major pathogenic features of H. capsulatum is its ability to establish a persistent state that is clinically unclear, with the exception of calcified granulomas observed on conventional radiographs of lungs, liver, or spleen [1].

Optimal host defenses against H. capsulatum require the interaction between macrophages and T cells. H. capsulatum replicates within macrophages until the T cells are activated. The signals that arm phagocytes to inhibit intracellular replication of H. capsulatum are not well understood. Peritoneal macrophages from mice are stimulated by IFN-γ, whereas other populations of macrophages are either unresponsive to IFN-γ or require a priming signal [2]. The only known activators of human macrophages are granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and macrophage CSF [3]. Of the many endogenous cytokines that may regulate immunity to pathogenic microbes, TNF-α appears to be the most influential in the development of a protective immune response against H. capsulatum [4–8]. Other cytokines that are implicated in the development of protective immunity against this fungus include IFN-γ, GM-CSF, IL-4, IL-10, and IL-12 [9–13]. The selection of TNF-α as perhaps the most influential cytokine for developing protective immunity against H. capsulatum is based on the necessity of this molecule for the development of protection against both primary and secondary histoplasmosis in animal models. No other cytokine has yet been reported to have the same degree of importance in host defenses. More recently, clinical reports have associated inhibition of the activity of endogenous TNF-α with the development of several infectious diseases, including progressive, disseminated histoplasmosis. Thus, TNF-α is a central mediator of resistance in both mice and humans [14, 15].

TNF-α And Primary Infection

After challenge with H. capsulatum yeast cells, there is a prompt and vigorous release of TNF-α [6]. In naive mice infected with H. capsulatum, neutralization of endogenous TNF-α by administration of either monoclonal antibody (MAb) or rabbit polyclonal antibody transforms a nonlethal infection into a lethal one. Death, which occurs at 7–20 days after infection, is associated with a markedly increased burden of H. capsulatum in both visceral and lymphoid organs [4, 8].

One possible explanation for the inability to control infection is the failure to generate levels of cytokines necessary for the elimination of H. capsulatum. Levels of IFN-γ and GM-CSF, 2 endogenous cytokines that regulate host resistance in primary infection, are not depressed in conjunction with neutralization of TNF-α. Moreover, the levels of IL-4, IL-10, or transforming growth factor–β, which are known inhibitors of cellular immunity, are not altered by neutralizing the biological activity of TNF-α in vivo [4, 8]. Another reason that H. capsulatum may grow unchecked in macrophages from TNF-α—neutralized mice is that a major signal for activation is not available. This argument is not supported by in vitro data showing that, after murine peritoneal or alveolar macrophages infected with H. capsulatum were exposed to recombinant TNF-α, the cytokine failed to directly trigger antifungal activity by the cells. To date, the only perturbation in immunity appears to be a decrement in the production of nitric oxide, which, in mice, is a key mediator of host resistance to this fungus. A deficiency of nitric oxide, observed either in nitric oxide synthase knockout mice or in mice given an inhibitor of this enzyme, results in the death of the mice. The underlying mechanism by which nitric oxide may contribute to immunity is by forming complexes with the essential nutrient iron and thus removing it from the fungus.

Another explanation for the failure of TNF-α—neutralized mice to control infection with H. capsulatum is modulation of the host inflammatory response. In H. capsulatum—infected mice administered TNF-α MAb, the number of inflammatory cells recovered from the bronchoalveolar lavage or the lungs was similar to that recovered from infected control mice. In addition, the architecture of the inflammatory response did not differ substantially between control mice and mice that received TNF-α MAb. The lungs of both groups of mice contained granulomatous inflammation of equal intensity [4].

The importance of TNF-α to host defenses is emphasized further in studies of histoplasmosis in IL-10 knockout mice. These mice are more resistant to primary challenge with H. capsulatum during the later stages of infection (day 7 and beyond) than are wild-type mice. However, the enhanced resistance is broached by neutralization of TNF-α in vivo. Among the perturbations detected in TNF-α—neutralized, IL-10 knockout mice is a decrement in the number of CD8+ cells in the lungs. Because these cells contribute to host defenses, the lower numbers may, in part, explain the depressed immunity. This alteration was not observed in wild-type mice administered TNF-α MAb. Thus, the absence of IL-10 in mice does not compensate for the lack of biologically active TNF-α in vivo [13].

The biological effects of TNF-α require binding to receptors present on the cell surface. TNF receptor (TNFR) 1 and TNFR2 transduce the signals that follow ligation and that mediate the downstream actions of TNF-α. In primary infection, the genetic absence of either TNFR1 or TNFR2 in mice is associated with a progressive course of infection after the mice are infected with 2 × 106 yeasts cells, and this course of infection is similar to that observed in mice that are TNF-α neutralized. However, there is a pronounced difference in susceptibility between the 2 knockout strains of mice. TNFR1 knockout mice die after challenge with as few as 103 yeast cells, whereas TNFR2 knockout mice are resistant to challenge with up to 105 yeast cells. This difference in resistance indicates that signaling through TNFR1 induces a more robust protective immune response than does signaling through TNFR2 [16].

Differences in the inflammatory and cytokine responses between the 2 knockout strains are apparent. In TNFR1 knockout mice, the number of inflammatory cells in the lungs is dramatically decreased, but levels of cytokines and nitric oxide are similar to those in control mice. In TNFR2 knockout mice, the inflammatory response to H. capsulatum is similar to that of infected control mice, but the levels of IFN-γ are lower than those in infected control mice and in TNFR1 knockout mice. The functional importance of this decrement to host resistance has been affirmed by administering recombinant IFN-γ to infected TNFR1 knockout mice and reversing the impaired immunity.

Another contrast worth noting is the response of the TNFR knockout mice, compared with that of mice administered TNF-α MAb. The only perturbation detected, to date, in the latter group of mice is diminished production of nitric oxide. Interestingly, this defect is not observed in either of the 2 knockout strains of mice. The production of nitric oxide by cells is mediated by signaling through one TNFR. These data suggest that the generation of nitric oxide would be impaired in mice deficient for both receptors.

TNF-α And Secondary Infection

Mice that have been immunized with viable H. capsulatum manifest accelerated clearance of secondary infection and can resist another lethal challenge. As in mice with primary infection, neutralization of TNF-α in H. capsulatum—immune mice abolishes the protective immune response [4, 8]. This finding has been observed in both wild-type mice and IFN-γ knockout mice treated with an antifungal to eliminate the fungus [8]. The high mortality rate among mice administered TNF-α MAb is not associated with marked perturbations in the inflammatory response, because the phenotype of cells infiltrating the lungs is similar to that of infected control mice [4]. In fact, the number of cells in TNF-α—neutralized mice is greater than that in control mice. Nitric oxide levels are similar between the 2 groups, but there is one sharp difference in the cytokine response. The lungs of mice administered TNF-α MAb manifest a striking increase in levels of both IL-4 and IL-10 between days 3 and 7 after infection. These findings suggest that the 2 cytokines influence the dysregulation of protective immunity, because both cytokines have been demonstrated to down-regulate the cellular immunity that is vital for host control of H. capsulatum. To prove this argument, mice were treated with TNF-α MAb and were administered IL-4 MAb or IL-10 MAb or both. The mortality rate among TNF-α—neutralized mice administered control antibody, IL-4 MAb, or IL-10 MAb was 100%, whereas the survival rate for mice administered both IL-4 MAb and IL-10 MAb approached 75% [4]. Suppression of the in vivo activity of elevated levels of IL-4 and IL-10 restores, to a large degree, the integrity of protective immunity and indicates that TNF-α neutralization in secondary histoplasmosis causes an enhancement in the signals that lead to excess production of both IL-4 and IL-10. Furthermore, the findings demonstrate that IL-4 and IL-10 must cooperate to depress cellular immunity.

A deficiency of IL-10 in mice is associated with a dramatic acceleration in the clearance of H. capsulatum. The difference between wild-type and knockout mice is even more pronounced in mice with secondary, rather than primary, infection. The disturbances in the inflammatory response are similar to those associated with primary infection (i.e., a reduction in the number of CD8+ cells). Interestingly, IL-4 levels are not dramatically elevated in IL-10 knockout mice administered TNF-α MAb, which suggests that up-regulation of IL-4, as seen in wild-type mice treated identically, must be dependent on the presence of IL-10 [13]. It remains to be determined whether the absence of IL-4 would affect the production of IL-10 in TNF-α—neutralized mice.

In secondary infection, TNFR1 is the mediator of resistance. Inhibition of signaling through this receptor profoundly decreases host resistance to a secondary challenge with H. capsulatum. On the other hand, the absence of TNFR2 does not affect the course of secondary infection. The increases in IL-4 and IL-10 levels that are observed in TNF-α—neutralized mice with secondary infection are mediated by signaling though TNFR1. Blockade of this receptor leads to an elevation of the levels of these cytokines in the lungs of mice. These findings clearly demonstrate that the inability to signal through TNFR1 is accompanied by an increase in both IL-10 and IL-4 levels. The elevation of the levels of these cytokines may be directly attributable to signaling through TNFR2, because it is the only receptor available for the endogenous TNF-α generated in secondary infection [16].

TNF-α And Regional Immunity

In mice with secondary histoplasmosis, the elimination of CD4+ cells, but not CD8+ cells, modestly impairs the clearance of H. capsulatum from the lungs and spleen, compared with that in control mice. This finding stands in sharp contrast to the absolute requirement for CD4+ cells in the control of primary infection [5]. Elimination of both CD4+ and CD8+ cells in mice with secondary murine histoplasmosis leads to the eventual death of the mice. However, the time until the mice die of infection is prolonged, compared with that noted for mice with primary infection. T cell—depleted mice with secondary infection do not begin to die of the infection until after day 30, whereas, for most mice with primary infection, death occurs between days 7 and 20 after infection [5]. The prolonged nature of survival suggests that in secondary infection, there are factors that can confer protective immunity to a limited degree in the absence of T cells.

Examination of the fungal burden in mice lacking both CD4+ and CD8+ cells demonstrated that the lungs controlled infection and even reduced the fungal burden, whereas there was an ∼4-log10 increase in the number of H. capsulatum colony-forming units. In mice lacking CD4+ cells, the number of colony-forming units decreased in both visceral and lymphoid organs. These findings were similar in B cell knockout mice, thus indicating that antibodies are not responsible for the effect observed in the lungs [5]. Analysis of the cytokine levels in mice lacking CD4+ cells and in mice lacking both CD4+ and CD8+ cells revealed that IFN-γ levels were markedly depressed in both groups of mice. Thus, IFN-γ could not account for the disparity in the survival rates of the 2 groups of mice. Because endogenous TNF-α is necessary for effective protection against secondary infection with H. capsulatum, it is possible that production of TNF-α by different organs is responsible for the divergent findings in visceral and lymphoid organs. Indeed, the lungs of T cell—depleted mice exposed to H. capsulatum can control the infectious process and can produce TNF-α in both T cell– and B cell—independent manners. On the other hand, the spleens of such mice fail to generate TNF-α [5]. The inability of the spleens of T cell—deficient mice to generate TNF-α provides an explanation for the progressive growth of the fungus in this particular organ system. Moreover, the findings indicate that the organs possess different mechanisms to combat infection but that TNF-α is a feature critical for all of them.

Human Studies

There is a paucity of information on how TNF-α blockade alters the human immune response to H. capsulatum. In vitro, TNF-α antibody or a control antibody modestly enhanced intracellular growth of yeast cells, and TNF-α MAb, but not control antibody, depressed proliferation of peripheral blood lymphocytes from healthy subjects in response to H. capsulatum. TNF-α MAbs also inhibited production of IFN-γ by either monocytes or alveolar macrophages from healthy subjects. Thus, in this study, TNF-α MAb caused a depression in T cell growth and production of a cytokine that may be important in human host defenses against H. capsulatum [15].

Summary

Histoplasmosis has been one of the principal infectious complications associated with inhibition of endogenous TNF-α. The mechanisms that underlie the increase in infectious complications are not fully defined. Depressed levels of nitric oxide and increases in IL-4 and IL-10 levels have been observed in wild-type mice with primary and secondary histoplasmosis, respectively. However, given the pleiotropic activities of TNF-α, it is unlikely that these are the only mechanisms. In human subjects, it appears as if there are alterations in the expansion of T cells and a decrease in IFN-γ levels, although the role that this cytokine plays in the control of histoplasmosis in humans has been identified. A better understanding of how TNF-α contributes to host resistance to H. capsulatum will serve to reduce the incidence of complications associated with its blockade.

Acknowledgments

I thank Mary Beth Poole for assistance.

Financial support. National Institutes of Health (grants AI-34361 and AI-42747).

Potential conflicts of interest. G.S.D., Jr. has been a consultant to and has received funding from Amgen.

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