Parasite Immunology, 2004, 26, 247264 Review Article

Hormonal and immunological mechanisms mediating sex differences in parasite in fection S. L. KLEIN The W. Harry Feinstone Department of Molecular Microbiology and Immunology, T he Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

SUMMARY The prevalence and intensity of infections caused by protozoa, nematodes, tremat odes, cestodes, and arthropods is higher in males than females. The primary thes is of this review is that immunological differences exist between the sexes tha t may underlie increased parasitism in males compared to females. Several field and laboratory studies link sex differences in immune function with circul ating steroid hormones; thus, the roles of sex steroids, including testosteron e, oestradiol, and progesterone, as well as glucocorticoids will be discussed. Not only can host hormones affect responses to infection, but para- sites can bo th produce and alter hormone concentrations in their hosts. The extent to which changes in endocrineimmune interactions following infection are mediated by the h ost or the parasite will be considered. Although males are more suscep- tible th an females to many parasites, there are parasites for which males are more resis tant than females and endocrine immune interactions may underlie this sex revers al. Finally, although immunological differences exist between the sexes, geneti c and behavioural differences may explain some variability in response to infect ion and will be explored as alternative hypotheses for how differences between the sexes contribute to dimorphic responses to parasites. Keywords cestode, oestrogen, glucocorticoid, innate immunity, nematode, protozo a, sex steroids, testosterone, Th1/Th2, trematode

Correspondence: Sabra L. Klein, Department of Molecular Microbiology and Immunol ogy, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA (e-mail: saklein@jhsph.edu). Received: 26 August 2004 Accepted for publication: 4 September 2004 INTRODUCTION Among human and non-human animals, the prevalence and intensity of parasitic infections is higher in males than females (Table 1; 13). Sex differences in exposure as well as susceptibility to parasites probably contribute to sexbased differences in the intensity and prevalence of parasites. For example, ma les are more likely to engage in behaviours, such as aggression, dispersal, and grouping, that increase the likeli- hood of contact with parasites (both ecto-

and endoparasites) (1,3). Males also often are larger than conspecific female s, which may make them more obvious targets for parasitism (4). Despite differ ences in the likelihood of exposure, several studies illustrate that immunologic al differences exist between the sexes that may underlie increased parasitis m in males. Females typically have higher immune responses than males (1,3). El evated immunity among females creates a double- edged sword, it which it is beneficial against infectious dis- eases, but is detrimental in terms of increa sed development of autoimmune diseases (5). Several field and laboratory studies link sex differences in immune function with circulating steroid hormones (13). Increased susceptibility to infection is thought to be one of the leading causes of increased death rates among men as compared with women (1,6). In a variety of host species, ranging from humans and rodents to birds a nd lizards, male-biased infection rates are most notable for protozoan and nemat ode parasites (Table 1). As illustrated in Table 1, of the 58 parasite speci es (proto- zoa, nematodes, trematodes, cestodes, and arthropods) for which s ex differences are reported, male-biased infection rates are observed for 49 (845%). Although males are more susceptible than females to many parasites, th ere are para- sites for which males are more resistant than females (7). Fo r example, male mice (Mus musculus) are less susceptible than females to sever al parasites, including Babesia microti, Toxoplasma gondii, Schistosoma mansoni , and Taenia crassiceps (811). The cause of reversed sex differences in response to 2004 Blackwell Publishing Ltd 247 S. L. Klein Parasite Immunology Table 1 Sex differences in the prevalence (P) and intensity (I) of parasite in fections in vertebrates Parasite Measure Sex difference Effect of hormones on susceptibility Protozoa Babesia microti I garis (8) Eimeria sciurorum P References Mus musculus E2 Host

F > M

Sciurus vul M > F (128) Entamoeba histolytica Homo sapiens I M > F (129) Giardia muris Mus musculus I M > F (61) Leishmania braziliensis Homo sapiens P M > F (23) L. donovani Homo sapiens P M > F (130) L. major Mus musculus I M > F T (27,69) L. mexicana Mus musculus

I cricetus auratus M > F erghei (69) L. guyanensis I

M > F (131) L. panamensis I

E2 Meso M > F

(131) Plasmodium b I (20) P. chabaudi Mus musculus I M > F T, C (1519,132) P. falciparum Ho mo sapiens I, P M > F (13,14,133) P. mexicanum Sceloporus occidentalis P M > F (134) Plas modium spp. Parus major P M > F (135) P. vivax Homo sapiens P M > F (136) Toxoplasma gondii Mu s musculus I F > M T, E2 (9,4042) Trypanosoma cruzi Mus musculus I M > F E2 (137,138) Calomys callosus I M > F T, E2 (139,140) Nematodes Angiostrongylus malaysiensis Rattus norvegicus I M > F T, C (79) Baylisascaris procyonis Procyon lotor I, P M > F (141) Brugia malayi Mu s musculus I M > F T (88) Meriones unguiculatus I M > F (142) B. pahangi Mus musculus I M > F T, C (143,144) Capillaria muris Clet hrionomys glareolus P M > F (145) Capillaria spp. Rattus norvegicus P M > F (146) Dirofilaria immitis Canis familiaris P M > F (147) Felis catus P M > F (148) Elaphostrongylus rang iferi Rangifer tarandus tarandus I M > F (124) Heterakis spumosa Mus musculus I M > F T, C ( 149) Muspicea borreli Mus musculus I, P M > F (150) Necator americanus Homo sap iens I M > F (151) Mesocricetus auratus I M > F (152)

Mesocricetus auratus T Mus musculus M > F T, C

Nematospiroides dubius Apodemus sylvaticus P M > F (153) Nippostrongylus brasiliensis Millardia meltada I M > F C (80) Onchocerca volvulus Homo s apiens I M > F (154) Rhabdias bufonis Rana temporaria P M > F E2 (155) Strongyloides ratti Mus musculus I M > F T, C (156) S. venezuelensis Rattus norvegicus I M > F C, O, T, E2 (157) Syphacia obvelata Apodemus sylvaticus P M > F (153) Mus musculus I, P M > F (158,159) S. stoma Apodemus sylvaticus I M > F (16 0) Trichinella spiralis Microtus pennsylvanicus I M > F (161) Rattus norvegicus I M > F T, C, E2 (162) Trichostrongylus retortaeformis Oryctolagus cuniculus I F > M O (163) Toxocara spp. Homo sapiens P M > F (164) Wuchereria bancrofti Homo sapiens P M > F (165) Trematodes Alaria taxideae Martes americana I M > F (166) Brachylaima cribbi Mus muscu lus I M > F (167) Crepidostomum farionis Salmo trutta I, P M > F (168) Discocotyle sagi ttata Salmo trutta I, P M > F (169) Gorgoderina vitelliloba Rana temporaria P M > F E2 (155) Polystoma integerrimum Rana temporaria P M > F E2 (155) 248 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 Volume 26, Number 6/7, June/July 2004 Sex diffe rences in parasite infection Table 1 Continued

Parasite Measure Sex difference Effect of hormones on susceptibility References

Host

Schistosoma mansoni Homo sapiens I, P M > F (30,31) Mus musculus I F > M T, C (10,39) Pseudodiplorchis americanus Scaphiopus couchii P M > F (170) Cestodes Echinococcus multilocularis Mus musculus I M > F (171) Hymenolepis nana Rattus norvegic us P F > M (146) Taenia crassiceps Mus musculus I F > M E2, T, C (11,44) T. solium Sus scrofa I F > M C (172) Arthropods Amblyomma hebraeum Tragelaphus strepsiceros I M > F (173) Centruroides limpidus limpidus Mus musculus I F > M (174) Hypoderma tarandi Rangifer tarandus tarandus I M > F C (81) Notoedres muris Rattus norvegicus P F > M (146) Polyplax spinulosa Rattus norvegicus P M > F (146) , not examined; T or , administration of testosterone increases or decreases infect ion in females, males, or both; C or , castration of males reduced or increases i nfection; E2 or , administration of oestradiol reduces or increases infection in females, males, or both; O or , ovariectomy of females increases or reduces infection. certain parasites is not well understood but may involve differences in ho stpathogen interactions that are affected by the endocrine system (7). The primary goals of this review are: (a) to illustrate that sex differences in response to parasitic infection are univer- sal (i.e. both across host and para site species); (b) to propose that sex differences in response to parasites a re caused by immunological differences between the sexes; and (c) to rev iew the possible mechanisms mediating sex differences in immune responses a gainst parasite infection. A second- ary goal of this review is to raise awar eness to the possibility that if males and females differ in their immunological responses to parasites, they may differ in their responses to treatments as we ll. Sex differences in the absorption, metab- olism, and overall effectivenes s of drug treatments are documented (5). The extent to which males and females differ in the immunogenicity of vaccines has not been well characterize d and may influence the effectiveness of parasite treatments.

SEX DIFFERENCES IN PARASITE INFECTION Field studies of vertebrates illustrate that the prevalence (i.e. proportion of individuals infected) and intensity (i.e. sever- ity of the infection) of para site infections are often higher in males than females (Table 1). The prevalenc e and intensity of infection with Leishmania, Plasmodium, Entamoeba, Necator, and Schistosoma parasites, for example, is higher among men than women (Tab le 1). Although clinical studies of humans and field studies of non-human a nimals are suggestive, several factors, including exposure rates, social behaviour, habitat, and diet cannot be held constant and could contribute to the observed differences in parasite infection. Studies of rodents in a controlled laboratory setting, however, reveal that sex differences are present and may be mediated by endocrineimmune interactions. Although males are more susceptible to a majority of parasite infections, exceptions are noted and should be f urther characterized (7). Male-biased infections One genus of protozoan parasites that causes a pronounced sexual dimorphism in vertebrate hosts is Plasmodium (Table 1). Among humans, although t he incidence of infec- tion is generally similar between the sexes (12, cf. 1 3), sex differences in the intensity of infection are reported in which men hav e higher parasitaemia than women (12,14). Studies of rodent malarias illustra te that mortality rates are higher in males than females and may involve immu nological differences between the sexes (Table 2; 15 19). Castration of males red uces, whereas exogenous administration of testo- sterone increases, mortality following infection with P. chabaudi or P. berghei in mice (18,20). In ad dition to increased mortality rates, male mice recover from P. chabaudi-indu ced weight loss, anaemia, and hypothermia slower and have lower antibody respo nses than females (21; A. Cernetich and S.L. Klein, unpublished data). Our pre liminary data also reveal that phenotypic differences between the sexes in response to P. chabaudi may be mediated by sex differences in the expressio n of genes that modulate pro-inflammatory and helper T-cell type 1 (Th1) re sponses (e.g. IFN) against infec- tion (A. Cernetich and S.L. Klein, unpublishe d data). 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 249 S. L. Klein Parasite Immunology Table 2 Examples of immunological differences between the sexes in response to parasite infection Parasite ales Response in males References Mice IFN Host Response in fem IgG1 DTH response

Leishmania major/mexicana (28,29) IgE IL-5 TNF Pl smodium chabaudi

Mice

IL

-2 mRNA IgG1 IFN mRNA MHC class II (17,19,132) Toxoplasma gondii IL-12 (9,42) IFN TNF Proliferation of splenocytes Schistosoma mansoni/haematobia (30,3336) IgG1 IgG2a IgG4 IgE IFN TNF Proliferation of PBMC

IgG

Mice

Humans IgA

IgG IL-10 TGF Cell death of PBMC

Taenia crassiceps IFN (46) IL-4 indicates elevated responses.

Mice IL-10

The immunomodulatory effects of testosterone may underlie increased susceptibili ty to Plasmodium infections in males compared with females. Exposure of a dult female mice to testosterone reduces antibody production, decreases major histocompatibility complex (MHC) class II cells in the spleen, and increase s CD8+ T-cells in the spleen (17). Th1 and Th2 responses are important for prot ection against infection with Plasmodium (22). Studies utilizing IFN (i.e. Th1) and IL-4 (i.e. Th2) knock-out (KO) mice reveal that the effects of IFN and IL-4 on survival are more pro- nounced in males than females. Male IFN and IL-4 KO mice have shorter survival times than their male wild-type (WT ) counterparts; in contrast, no differences are observed between female KO and WT mice (19). Sex differences are apparent in both IFN and IL-4 KO mice in which mortality rates are still higher in males than females (19). Epidemiological studies of Leishmania infections reveal that adult men ar e more frequently infected than women and that sex differences in behaviou r (e.g. via occupational exposure) as well as host immune responses to infectio n are involved (2325). Sex differences in response to Leishmania infection are re ported in pre-pubertal children, in which boys are more likely to develop visce ral leishmaniasis than girls (26). Whether sex differences in the prevalence o f leishmaniasis are caused by behavioural risk factors or by differences in endocrineimmune interactions requires further investiga- tion. The extent to w hich sex steroids alter the development of the immune system during the critic al period of sex differ- entiation to influence responses to infection prior t o puberty and into adulthood should be considered (1). Experimental studies of Leishmania infection in mice also reveal that mal es are more susceptible to infection than females (Table 2). Castration of mal es reduces, whereas administration of testosterone to females increases, suscep-

tibility to L. major (27). Males also are more susceptible than females to infe ction with L. mexicana and this sex difference appears to be mediated by the e ffects of oestrogens on the synthesis of IFN and production of Th1 responses (2, 28,29). Among children and adults, the intensity and prevalence of Schistosoma infection in endemic areas is higher in males than females (30, 31). Sex differences in the prevalence of infection may be attributed to diff erences in the amount of time spent in water and, hence, exposure to snails (i.e. the intermediate host) or differences in skin lipids that may infl uence the ability of Schistosoma parasites to penetrate skin and cause in fection (32). Increased parasite burden in males, however, is hypothesized to contribute to the elevated pro-inflammatory (e.g. TNF), Th1 (e.g. IFN), Th2 (e .g. IgE), and antibody responses in males compared with females (Table 2; 3336). Consequently, elevated pro-inflammatory 250 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 Volume 26, Number 6/7, June/July 2004 Sex diffe rences in parasite infection responses against S. mansoni are correlated with develop- ment of disease s, including hepatosplenomegaly (37). In contrast, regulatory T-cell response s, including the synthesis of IL-10 and TGF, that down-regulate Th1 responses, a re higher in females than males (35). IgA production, which provides immunity against reinfection with Schistosoma parasites, also is higher in females than males (35). Whether hormones mediate sex differences in responses to Schistosoma parasites in humans is unclear; oestrogens and progestins, however, are hy pothesized to regulate elevated IL-10, TGF, and IgA production in females (35). Studies of Schistosoma infection in humans represent one of the few hostparasit e systems for which sex differences in immune responses are well characterized (Table 2) and should serve as a model for studies of other parasites that exhib it sexually dimorphic patterns of infection in humans. Mouse models of S. mansoni infection are difficult to interpret in relat ion to human studies because the sex differ- ence is reversed, in which female m ice are more susceptible to infection and develop higher pro-inflammatory immu ne responses than males (10,38). Administration of testosterone protects, where as castration exacerbates worm burden and death following inoculation with S. mansoni parasites (39). Why the sex difference in response to Schistosoma para sites is reversed in mice compared with humans has not been resolved and r equires additional investigation. Female-biased infections One of the best studied parasites for which females are more susceptible than ma les is Toxoplasma gondii. In mouse models, females develop more severe brain in flammation and are more likely to die following infection than males (9). Ova - riectomy of female mice reduces, whereas administration of oestradiol exacerb ates, the development of tissue cysts caused by T. gondii infection (40,41). Male mice produce higher concentrations of TNF, IL-12, and IFN than fema les early during infection (Table 2) (9,42). Human studies of sex differences in T. gondii infection are scarce because most healthy adults are asymptomatic. Among immunocompro- mised individuals, however, T. gondii-induced encephalitis is more prevalent among women than men (43). Taenia crassiceps is an intestinal cestode for which rodents serve as an interm ediate host. Studies of mice reveal that females develop more cysticerci than males (11). Oestrogens favour, whereas androgens inhibit, T. crassiceps grow th and development (44,45). Males develop higher Th1 responses, including elev ated IFN synthesis, whereas females exhibit heightened IL-10 production, during

the early phase of infec- tion (Table 2) (46). Because Th1 responses inhibit p arasite growth, this is hypothesized to be the proximate mechanism mediating reduced susceptibility to infection in males (46). IMMUNOLOGICAL DIFFERENCES BETWEEN THE SEXES Sex differences in immune function are well established in vertebrates (1,2, 47). Males generally exhibit lower immune responses than female conspecifics (1, 2,47). Specifically, innate responses, antibody-mediated responses, and cellular responses typically are higher in females than males. Immunological differe nces between the sexes may explain why males and females differ in their responses to parasites. Innate immunity Innate immunity represents the first line of defence against parasites. Bec ause these responses do not require prior exposure or sensitization, the y can be initiated immediately following exposure to a novel parasite. Males and females differ in their innate immune responses, suggesting that som e sex differences may be germline encoded. Studies of both humans and rodents illustrate that inflammatory immune responses are generally higher in female s than males and may explain why women are more likely to develop inflam- mato ry rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythem atosus, than men (48). Female mice also exhibit stronger delayed-type hypers ensitivity reactions to Schistosoma mansoni than males (38). Following antigeni c stimulation, males produce higher concentrations of the inflammatory mediator, bradykinin, than females and this response is mediated by sex steroid hormo nes (49). The number and activity of cells associated with innate immunity differ bet ween the sexes. Phagocytic cells, including macrophages and neutrophils, can kil l parasites by generat- ing reactive oxygen metabolites and nitric oxide, as we ll as by secreting enzymes. Among humans and lizards, the phago- cytic activit y of neutrophils and macrophages is higher in females than males (50,51). Fo llowing parasitic or antigenic stimulation, the production and release of prost aglandin E2, thromboxane B2, and nitric oxide is reportedly higher in females than males (50,52,53). Other studies, however, demonstrate that plasma concentrations of several pro-inflammatory cytokines, includ ing IL-6 and TNF, are higher in males following trauma (54). Natural killer (NK) cells also repre- sent a critical first line of defence against parasites . Women with regular menstrual cycles as well as women tested dur- ing the lu teal phase of their menstrual cycle have lower NK cell activity than men (55,5 6). Studies of mice illustrate that oestradiol can reduce both the number and ac tivity of NK cells (57). Antigen-presenting cells (APC) from females are more ef ficient at presenting peptides than are APC from males (58). Following infection of the central nervous system, the expres- sion of MHC class II on astrocytes , endothelial cells, and microglia is enhanced in female compared with male mi ce (52). 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 251 S. L. Klein Parasite Immunology Although these data illustrate that innate immunity dif- fers between the s exes, whether the sexes differ in their reli- ance on innate immunity to ov

ercome parasitic infections has not been well documented and represents an im portant area for future research. Immunodeficient mouse models may be util ized to differentiate the roles of innate and acquired immunity in mediating sex differences in response to infection. For example, in response to Toxoplasma go ndii, both female WT and SCID (T- and B-cell deficient) mice are more sus- cept ible to infection (i.e. develop larger lesions and die faster) than the ir male counterparts, suggesting that the sex difference is mediated by inna te and not acquired immune responses against infection (9,42). Male SCID mice have significantly higher plasma concentrations of IL-12 and IFN, suggesti ng that sex differences in the responses of macro- phages and NK cells may mediate sex differences in suscep- tibility to T. gondii (9). Whether the sexes differ in their reliance on subsets of immune cells for recovery from other para sites has not been adequately studied and requires additional investigation. Acquired immunity Humoral immune responses (i.e. antibody production by B-cells) are typically greater in females than males (59,60). In mice infected with the parasite Giar dia muris, females have lower infection rates and higher antibody production than males, suggesting a functional advantage for elevated humoral immunity i n females (61). Cell-mediated immune responses also differ between males and females. T-cells, in particular CD4 + helper T-cells (Th cells), are functionally and phenotyp ically heterogeneous and can be differentiated based on the cytokines they rele ase. Reliance on subsets of Th cells (i.e. Th1 or Th2 cells) to overcome infection differs between males and females, with females reportedly exhibit ing higher Th2 responses (i.e. higher IL-4, IL-5, IL-6, and IL-10 producti on) than males (2,62). There are reports of females having higher Th1 responses (i.e. higher concentrations of IFN) than males (63,64). Female rodents also have higher mitogen-stimulated lymphocyte proliferation, faster wound healing, and increased immunological intolerance to foreign substances than males (65 6 7). Sex differences in Th cell responses may mediate sex dif- ferences in response t o infection. After experimental inocu- lation with coxsackievirus, male mic e primarily generate Th1 phenotypic responses (i.e. elevated IFN, IL-2, IgG2 a) and females exhibit predominantly Th2 phenotypic responses (i.e. high IL-4, IL-5, IgG1) (68). Treatment of males with oestradiol and females with testos terone prior to coxsackie- virus infection reverses the Th responses (68). Femal e DBA / 2 mice are more resistant to cutaneous Leishmania mexicana infection than males (69). In this case, females produce higher IFN responses a gainst L. mexicana and ovariectomy of female mice suppresses IFN synthesi s (69). Similarly, in humans, women generate a more robust delayed-type hyper - sensitivity response (i.e. Th1-related), whereas men have higher IgE con centrations (i.e. Th2-related) following infected with L. mexicana (25). Female mice are more susceptible to T. gondii infection and tend to have lower IL12 and IFN responses than males (9). Conversely, females produce higher concentr ations of IL-10, which could antagonize IFN responses against T. gondii (42). Taken together, these data illustrate that sex differences in cytokine respons es to infection play a critical role in determining susceptibility to parasites . ENDOCRINE-IMMUNE INTERACTIONS AND RESPONSES AGAINST PARASITIC INFECTION The prevailing hypothesis for immunological differences between the sexes is tha t sex hormones, in particular, testo- sterone, 17-oestr diol, and progesterone, influence the immune system. The localization of sex hormone receptors in immun

e cells, including lymphocytes, macrophages, granulocytes, and mast cell s, illustrates that there are direct connections between the endocrine and i mmune systems and that endo- crine factors can directly modulate the expressi on of target genes in immune cells. Thus, considerable research has focused on u ncovering sex steroid hormone actions in the immune system and how these influence sex differences in healthy and diseased conditions. Although sex differences in the synthesis and release of sex steroids are hypothesize d to underlie discrepancies in responses to infection, it remains unclear whether sex steroids explain all sexual variation in responses to infection (1). Androgens Sex differences in infection are mediated, in part, by the effects of and rogens, including dihydrotestosterone (DHT) and testosterone, on the immune system (Table 3) (2,70). Androgen receptors have been identified in various l ymphoid tissues, including the thymus, bone marrow, and spleen of rodents, as well as in primary cultures of macrophages (2,71,72). Exposure to testostero ne, the primary biologically active androgen, in vivo reduces NK cell activit y in mice (73). Similarly, stimulation of murine macrophages with testo- steron e in vitro reduces the synthesis of pro-inflammatory products, including TNF and nitric oxide synthase (74). In contrast, testosterone increases synthesis of anti-inflammatory cytokines, such as IL-10 (74). The immunosuppressive effects of testosterone may reflect the inhibitory effects of androgen receptor signall ing mechanisms on transcriptional factors (e.g. NF-B) that mediate the product ion of pro-inflammatory 252 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 Volume 26, Number 6/7, June/July 2004 Sex diffe rences in parasite infection Table 3 Effects of steroid hormones on the immune system Hormone gical outcome References DHT IL-1, IL-6 production (175) IFN production (63) IL-4, IL-5 production (63) IL-4 mRNA (176) CD4+ T-cells (176) Antibody production (177) Fc receptor expression (178) Testosterone M numbers (179) Nitrite production by M (91) Immunolo

 iNOS mRNA (180) Eosinophil degranulation (181) TGF (182,183) TNF production (184) IL-4, IL-5 (182) IL-4, IL-5, IL-10, IL-13 production (74,185,186) IFN mRNA and production (182,187) Bcl2 activity (188) CD4+ and CD8+ T-cells (189) CD4+ T-cells (190) Antibody production (191) Fc receptor expression (178) Estradiol M phagocytosis (192) Nitrite production by M (91) Eosinophil degranulation (181) NK activity (83,193) NK activity (57,194) MAPK activity (195) NF-B activity (86) TGF (196) IL-1, IL-6, and TNF production (70,74,184,197) IL-1, IL-6, and TNF production (198201) IFN mRNA and production (202) IFN production (203) TNF production (198) IL-2 mRNA (202) IL-2 production (186,203) IL-4, IL-5, IL-10, IL-13 mRNA / production (185,186,203) T-cell apoptosis (204) Bcl2 activity (188)

 CD8+ T-cells (205) Lymphocyte activation markers (206) (CD40, CD44, CD69, IL-2R) B cell activity (207,208) Antibody production (60,98,207,208) Fc receptor expression (209) Wound healing (196) Progesterone Nitrite production by M (91) NO production by M (84,95) Eosinophil degranulation (181) NK activity (9294) NF-B production (96) TNF mRNA, and production (95,96) TNF production (184) IL-2 mRNA (186) IL-4, IL-5 production (97,186,210,211) IgA and IgG production (98) CD30 expression (97) 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 253 S. L. Klein Parasite Immunology Table 3 Continued Hormone gical outcome References Cortisol / Corticosterone (212) iNOS mRNA (213) NK activity (214) NF-B activity (215) TGF production (210) NOS activity Immunolo

 IL-1, IL-6, and TNF production (100,210,216,217) IL-12 activity (99,218,219) STAT-4 activity (218) IFN, IL-2, and TNF production (210,220222) IL-4 and IL-10 production (210,219221) GM-CSF (223) lymphocyte & monocyte apoptosis (102,104,224,225) DTH (103) Circulating lymphocytes & monocytes (226) Antibody production (227,228) CD8+ T-cells (229) Lymphocyte proliferation (230) indicates that the hormone increases the listed response; indicates that the hormone decreases the listed response. and anti-parasitic cytokines (75). Alternatively, androgens may suppress immun e responses by increasing the expression and translation of stress proteins (e.g. heat shock proteins) and apoptosis factors (76 78). Gonadectomized male mice show greater resistance to several protozoan paras ites, including Leishmania major, Plasmodium berghei, and P. chabaudi, as compared with gonadally intact male mice (1520,27). In response to Angi- ostro ngylus malaysiensis infection, gonadectomized male rats (Rattus norvegicu s) have reduced numbers of worms, increased numbers of circulating leuco cytes, and heavier thymic mass than gonadectomized males injected with te stosterone propionate (79). Infection of Indian soft-furred rats (Millardia me ltada) with the parasite Nippostrongylus brasiliensis results in a higher worm burden in gonadally intact males than in females or castrated male rats (80). Gonadally intact male reindeer (Rangifer t. tarandus) show higher incidence of warble fly (Hypoderma tarandi) infesta- tion than both females and castrated males (81). Although androgens can modify responses to infection, the mechanisms mediat ing this effect often are unclear. For example, the effects of testosterone on P. chabaudi infection are not mediated by traditional ligandreceptor intera ctions because pharmacologically blocking either intracellular androgen or oest rogen receptors in adulthood has no effect on infection (15,16). Thus, sex differences in P. chabaudi infection cannot solely be explained by the direc t actions of circulating sex steroid hormones in adulthood, suggesting that other mechanisms are involved. Testosterone may have non-genomic effects on mal aria infection by binding to nontraditional membrane receptors coupled to G proteins on the surface of T-cells and macrophages (72). Alternatively, the effects of test osterone on responses to Plasmodium infec- tion may be organized by the presence or absence of andro- gens earlier during development. Oestrogens

Oestrogens modulate immune function in females and may contribute to resistan ce against infection (Table 3). Oestrogens affect both innate and acquired imm une function (Table 3). Oestrogen receptors are expressed in various lymphoid t issue cells as well as in circulating lymphocytes and macrophages (2,71,82). Ex posure of human NK cells to 17-oestr diol in vitro enhances NK cytotoxicity (83 ). Oestrogens also stimu- late synthesis of pro-inflammatory cytokines, incl uding IL-1, IL-6, and TNF (84). Oestrogens can enhance both cell-mediated and h umoral immune responses; there are, however, reports of oestrogens suppressing s ome cell-mediated immune responses (85). The cellular and molecular mech- a nisms mediating oestrogenic effects on immune function have not been fully elucidated. The effects of oestrogens on transcriptional factors, such as NF -B, are cell-specific in which oestrogens either enhance or inhibit NF-B signall ing pathways, depending on the cell type (86,87). Oestrogens also may enhan ce immune function in females by protecting immune cells against apoptosis (76, 78). Female resistance to infections caused by parasites, includ- ing Brugia malayi and Leishmania mexicana, is positively associated with oestrogen concentratio ns (69,88). Resistance against L. mexicana is related to the effects of oestroge n on increased transcription of IFN mRNA in mice (89). If females 254 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 Volume 26, Number 6/7, June/July 2004 Sex diffe rences in parasite infection are depleted of IFN using monoclonal antibodies or if males are administered rec ombinant IFN, then sex differences in disease progression can be reversed (89). The effects of oestro- gens on responses to infection may be hard-wired early du ring ontogeny. For example, although females are more resistant to Brugia malay i infection than males, gonadectomy of adult females has no effect on resistance to infection; conversely, gonadectomy prior to puberty increases suscept ibility to B. malayi in adult females, suggesting that exposure to sex ster oids early during development may influence the expres- sion of sex differences in parasite infection later in life (88). Adult females are not resistant to all infections; female mice treated with pharmacological doses of 17-oestr diol are more susceptible to Toxoplasma gondii than non-treated females or males (40). Increased susceptibility to T. gondii in oestrogen-treated mice may be due to reduced innate immunity (e.g. NK cell and macrophage activity) an d lower cytokine responses (e.g. IL-12 and IFN production) (9,40). These data s uggest that the effect of endocrineimmune interactions on responses to infec tion may be parasite-specific. Whether changes in host endocrine and immune re sponses following infection are mediated by the host or by the parasite must be considered in order to fully elucidate the nature of these dynamic relationships. Progestins Progestins, primarily progesterone, play a critical role in reproduction, including the maintenance of pregnancy in mammals, and immune function. Pro gesterone can have both stimulatory and suppressive effects on the immune sys tem, but is typically regarded as immunosuppressive (Table 3). Pro- gesterone r eceptors have been identified in epithelial cells, mast cells, granulocytes (e.g. eosinophils), macrophages, and lymphocytes (2,84,90). Progesterone can bind to glucocorti- coid receptors, which are more abundant in the immune system than progesterone receptors, and may represent an alternative mecha

nism for progesterone-induced changes in immune function (84). Progesterone suppresses innate immune responses, including macrophage and NK c ell activity as well as NF-B signal transduction (75,84,9194). Progesterone can inhibit nitrite and nitric oxide production as well as TNF mRNA and productio n by murine macrophages (91,95,96). Elevated concentrations of progesterone duri ng pregnancy inhibit the development of Th1 immune responses that can lead to f oetal rejection and promote production of Th2 immune responses, including IL-4 and IL-5 production (90,97). Thus, proges- terone is considered to be an an ti-inflammatory hormone. Progesterone also suppresses antibody production, which may be caused by progesterone inhibiting CD8+ T-cell responses that in turn suppress antibody production by B-cells (98). Although the immunomodulatory properties of progesterone are well characterized in both mice and humans, the effects on responses to infecti on have not been ade- quately examined and may contribute to sex differen ces in response to parasitic infections. Glucocorticoids Glucocorticoids, including corticosterone in rodents and cortisol in primates, may influence sexual dimorphisms in immune function. Glucocorticoid receptors have been identified throughout the immune system and in circulating T-cells, Bcells, and macrophages (99,100). High circulating concentrations of corticoster one suppress innate (i.e. NK activity), cell-mediated (i.e. cytokine productio n), and humoral (i.e. antibody production) immune responses in laboratory rat s and mice (Table 3) (99,100). Generally, glucocorticoids suppress the synthesi s of pro-inflammatory cytokines, including IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, TNF, IFN, and GM-CSF, and enhance production of anti-inflammatory cytokines, such as IL-4 and IL-10 (100). Glucocorticoids alter production of many cytokines by affecting production of prostaglandins and nitric oxide (100). Adrenalectomy increases lymphoid tissue mass and B-cell activity in mice (101). The immuno suppressive effects of glucocorticoids are mediated by the antagonistic effec ts on NF-B-medi ted responses (75). Glucocorticoids also cause lymphocyte apopt osis and redistribution of lymphocytes from blood to organs, including ly mph nodes, skin, and bone marrow (102104). Laboratory studies in rodents illustrate that basal corti- costerone concen trations are higher among females and rise more rapidly in females than males following exposure to stressors (105,106). Males and females often diffe r in the amount of stress they experience and the types of stressors they enco unter. Presumably, if infection disrupts homeosta- sis differentially between males and females, then the effects of glucocorticoids on immune responses may d iffer between the sexes. The role of glucocorticoids as mediators of sex dif- fe rences in susceptibility to parasitic infection has not been adequately explo red and represents an important area of future research. Androgens and oestrogens have been the primary focus of studies examining sex d ifferences in infection. Several other steroid hormones, including progestins , glucocorticoids, as well as pituitary-derived peptide hormones, such as prol actin and growth-hormone, exhibit sexually dimorphic patterns of expression a nd may influence sex differences in infection. Because peptide hormones are di rectly affected by steroid hormone concentrations via feedback mechanism s, it is equally likely that sex differences in infection represent the interplay between peptide and steroid hormones (7). Thus, 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 255 S. L. Klein Parasite Immunology

the endocrine system may mediate sex differences in infection through a variety of mechanistic pathways. Parasites alter host hormone concentrations Not only can host hormones affect responses to infection, but parasites can have pronounced effects on hormone sig- nalling within the host. Male mice ar e more susceptible to Plasmodium berghei and P. chabaudi than females (18,20 ). Although several studies illustrate that host sex steroids influence the cour se of Plasmodium infection, additional studies suggest that these protozoan para sites can alter hormone concentra- tions in their hosts. Infection of female mi ce with P. berghei increases concentrations of oestradiol and progesterone and disrupts oestrus cyclicity (107,108). Infection with P. chabaudi suppresses test osterone concentrations in males (109). Whether these hormonal changes following infection are mediated by the parasite or host remains elusive. Host castratio n by para- sites is reported in both vertebrate and invertebrate hosts and is hypothesized to increase the availability of host resources for parasite grow th and development (110). Schistosoma mansoni infection, for example, suppres ses testosterone pro- duction in male mice (111). Because elevated oestrogen a nd suppressed androgen concentrations can facilitate immune responses against Plasmodium infection, these hormonal changes may be adaptive host responses aga inst infection as opposed to examples of parasite-mediated castration. Female mice are more susceptible to infection with the tapeworms Taenia crassiceps and T. taeniaeformis than males because oestradiol enhances p arasite reproduction (11,112,113). Infection of male rodents with T. crassiceps or T. taeniaeformis reduces both serum and testicular testoster- one concentrati ons, increases oestradiol concentrations, and inhibits mating behaviour (11,112 ,113). Taenia crassiceps parasites also produce steroid hormones, including test o- sterone (114). Because testosterone can be aromatized into oestradiol in th e host, testosterone production by the para- site may further facilitate growt h and reproduction of the parasite and inhibit host responses to infection ( 114). Taken together, these data illustrate that the effects of parasites on h ost hormones may contribute to the observed (and often unexplained) variabi lity in the expression of sex differences in response to different parasite sp ecies. ADDITIONAL FACTORS THAT MAY INFLUENCE SEX DIFFERENCES IN PARASITE INFECTION Genes Several studies illustrate that host genes play a critical role in mediating susceptibility and resistance to parasites. Resistance to P. chabaudi is polygenic and several loci have been identified that influence susceptibility to infection (termed Char 1 4, for Chabaudi resistance). Linkage ana- lyses have mapped these loci to chromosomes 9, 8, 1 7, and 3, respectively. Crosses between resistant and susceptible strains, which result in recombinant inbred strains, reveal that the effects of Char2 and Char4 on resistance to infection are more pronounced among females than males (1 15,116). Thus, genetic resistance to P. chabaudi is sex-dependent, although the role of sex steroids has not been reported. Genetic resistance to L. mexicana has been mapped to a single locus, Scl-2, th at is located on chromosome 4 and that mediates a no lesion growth phenotype (11 7). Studies of backcross and F2 recombinant inbred strains of mice (from parent al C57BL/6 and DBA /2 mice) reveal that the effect of Scl-2 on resistance to L. mexicana is more pronounced among female than male mice (117). The genes t hat encode for Janus tyrosine kinases (JAK)-1 and -2 have been mapped in

to the same region on chromosome 4, which suggests that Scl-2 may play a role in cytokine-mediated pathways that differ between the sexes (118). Like P. chabaudi and L. mexicana, susceptibility to mousepox has been mapped to disease-related loci on autosomal chro- mosomes. Using recombinant inbred s trains of mice, four loci have been identified, Rmp1 4 (resistance to mousepox loci), that confer resistance to mousepox. The effects of loci Rmp2 (on chromoso me 2) and Rmp4 (on chromosome 1) on resistance to infection differ between the sexes, such that these loci confer greater resistance in female than male con- genic mice. If congenic mice are neonatally gonadectomized at 47 days of a ge and infected with mousepox as adults, then the sex difference in resistanc e is abolished; gonadect- omized males and females are equally susceptible to m ouse- pox (119). Neonatal ovariectomy increases female susceptibility to mousep ox, whereas castration of neonatal males has little effect on susceptibility t o infection (i.e. castrated males are as susceptible as intact males) (119). Thus, oestrogens may enhance as opposed to androgens suppressing genetic resist ance to mousepox. These data also indicate that early hormonal manipulation may have profound effects on the expression of disease resistance genes in adult hood. Before the precise effects of sex steroid hormones on disease resist- anc e genes can be delineated, studies must consider the role of sex steroids durin g critical periods of sex differentiation. Behaviour In addition to immunological and genetic differences between males and females , behavioural factors can modify the expression of sex differences in infection. Behavioural vari- ation between the sexes can result in differential expos ure and contact with parasites. Sex steroid hormones regulate 256 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 Volume 26, Number 6/7, June/July 2004 Sex diffe rences in parasite infection several behaviours, such as aggression and reproduction, that are related to th e spread of infection. Males of several species often are more aggressive than females and this beh avioural response is mediated, in part, by circulating androgens. In males, high testosterone con- centrations enhance competition and aggressiveness and in crease susceptibility to parasites (3,120,121). Despite the advantages of hig h testosterone concentrations on reproduc- tive success, testosterone-dependent aggression (i.e. inter-male aggression) increases exposure to parasites and may underlie the increased prevalence of infections among males as com- pared with f emales (121). In addition to increasing exposure to parasites, several laborat ory studies suggest that engaging in aggressive acts can increase androgen conc entrations and predispose males to be more susceptible to infection than f emales. Specifically, studies of mice illustrate that high- ranking, dominant ma les engage in more aggressive encoun- ters and are more susceptible to infect ion with nematodes, such as Heligmosomoides polygyrus, and protozoa, including Babesia microti, than less aggressive subordinate males (122,123). Reduced clear ance of infection among high ranked males is associated with high testosterone and corticoster- one concentrations, suggesting that steroids in addition t o testosterone mediate sex differences in infection (122,123). Different reproductive strategies employed by males and females can modify pa rasite exposure and susceptibility to infection. Sex steroid hormones are directly related to the expression of characteristics (morphological and behav- ioural) important for breeding (3). Sex steroid hormones are hypothesiz ed to create a trade-off for males between the beneficial effects of an

drogens on the expression of secondary sex traits and the detrimental effects of androgens on sus- ceptibility to infection (3,4,121). Consequently, several studies suggest that males are more susceptible to infections during the breed ing than non-breeding seasons. For example, male reindeer are more suscepti ble to parasite infections during the rut season than during other times of t he year (81,124). Reproductive and aggressive behaviours may increase stressor e xposure in males as compared to females and may increase male susceptibility t o infection, at least during the breeding season. The role of stress and stress-related hormones as mediators of sex differences in parasite infec tion requires additional investigation. The extent to which behavioural risk f actors associated with occupation and extracurricular activ- ities differ betwee n the sexes should continue to be explored. Finally, whether behavioural differe nces between the sexes that influence contact with common vectors, such as m os- quitoes, contribute to sex differences in the prevalence and intensity of vector-borne parasitic infections should be exam- ined. Alternatively, sex diff erences in the prevalence of vector- borne parasites may be caused by different ial preference of the vector for a particular host. For example, some species of Mansonia mosquitoes prefer to feed on female rather than male human hosts ( 125). DISCUSSION/FUTURE DIRECTIONS The sexes differ in their responses to infection. The intensity and prevalence o f parasite infections typically are higher in males than females. Endocrineimmu ne interactions play a fundamental role in mediating responses to infection. Bec ause sex steroid concentrations differ dramatically between the sexes, to dat e, most studies have focused on characterizing the role of sex steroids as me diators of sex differences in parasitic infection. Future studies must contin ue to examine whether other steroid and peptide hormones contribute to sex diffe rences in parasite infection. Additionally, many studies characterizing the eff ects of hormones on the immune system use in vitro tissue culture systems (7). Whether natural hor- monal fluctuations associated with puberty, pregnancy, or menstruation affect responses to parasites should be further examined. Fi nally, whether changes in host endocrine and immune responses following i nfection are mediated by the host or by the parasite must be considered, to fu lly elucidate the nature of these dynamic relationships. The immune systems of males and females differ. Most studies characterizing sex differences in immune responses to infection focus on acquired immune resp onses, with par- ticular attention paid to the Th1 / Th2 dichotomy. Although th is approach has yielded valuable information about the causes of sex differe nces in parasite infection (see Table 2), future studies should move beyond th is paradigm and begin to consider other host responses to infection. For many p arasites, absolute concentrations of cytokines do not defin- itively predict su sceptibility or resistance to infection. Rather, the timing of cytokine produc tion as well as proper regula- tion of immune responses by regulatory T-cells may play a key role in maintaining immunity against parasites. Although sex di fferences in the synthesis of regulatory cytokines, such as TGF, have been re ported (35), future studies should continue to characterize sex difference s in regulatory T-cell pathways. Responses of the innate immune system pl ay a critical role in the initial recognition and response to parasites and m ay alter the expression of sex differences in parasite infection. Pattern rec ognition receptors, such as toll-like receptors (TLR) in vertebrates, are int imately involved in mediating host innate responses to infection and serve a s a bridge between innate and acquired immunity. Whether the sexes differ in the expression of TLR, mannose receptors, or scavenger receptors that bind to an d mediate internalization of parasitic particles has not been reported, but coul d influ- ence dimorphic responses to infection. The functional significance of sex differences in immune

responses against infection must be considered. For example, 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264 257 S. L. Klein Parasite Immunology do males and females differ in their responses to treatments, such as vaccines, against parasites? Studies of both humans and mice reveal that the sexes differ in their responses to vaccines and commonly used vaccine antigens. For example, protection by vaccines developed against P. chabaudi is greater for females tha n males and elevated testosterone concentra- tions reduces the efficacy of vacci nes against P. chabaudi in mice (126). Although the prevalence and intensity of Schis- tosoma worms is higher in men than women, men develop higher antibody r esponses against the worms and surface antigens (e.g. Sh28GST) than women (35,36 ). Consequently, testosterone binds to the Schistosoma glutathione S- transferas e (GST) protein with high affinity, and may modu- late host responses to the pa rasite and to vaccines developed against Schistosoma surface antigens (127). Th e extent to which endocrineimmune interactions affect the immunogenicity of vac cines should be considered. Taken together, the data presented in this review illustrate that parasites dif ferentially affect males and females, and interactions between the endocrine and immune systems may mediate sex differences in response to infection. This growi ng field relies on knowledge and expertise from parasitology, immunology, e ndocrinology, and related subdisciplines. Cross- talk among these disciplines wi ll be instrumental for this inte- grative field to move beyond characterization of dimorphisms in infection and into demonstrating the specific mechanisms that mediate sex differences in response to parasites. ACKNOWLEDGEMENTS I thank Alan Scott, Randy Nelson, and two anonymous referees for helpful suggestions on earlier drafts of this manuscript. I also thank the parti cipants of the Sexual and Immune Dimorphism in Parasitic Diseases Workshop, he ld in Cuernavaca, Mexico, for increasing my knowledge about the generalities as well as the complexities associated with sex differences in response to paras ites. Support for the data from my laboratory was provided by a grant from the Johns Hopkins Malaria Research Institute (SLK). REFERENCES 1 Klein SL. The effects of hormones on sex differences in infec- tion: from ge nes to behavior. Neurosci Biobehav Rev 2000; 24: 627 638. 2 Roberts CW, Walker W & Alexander J. Sex-associated hor- mones and immu nity to protozoan parasites. Clin Microbiol Rev 2001; 14 (3): 476 488. 3 Zuk M & McKean KA. Sex differences in parasite infections: patterns and processes. Int J Parasitol 1996; 26: 10091023. 4 Moore SL & Wilson K. Parasites as a viability cost of sexual selection in natural populations of mammals. Science 2002; 297 (5589): 2015 2018. 5 Wizemann TM, Pardue M, eds. Exploring the Biological Con- tributions to Hu man Health: Does Sex Matter?. Washington DC: National Academy Press, 2001. 6 Owens IP. Ecology and evolution. Sex differences in mortality rate. Science 2002; 297 (5589): 2008 2009.

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264 2004 Blackwell Publishing Ltd, Parasite Immunology, 26, 247264