Natural and controlled experiments provide evidence in favor of testosterone-mediated male survival, with castrated males living longer than intact males across many species including humans .Immune activation increases resting metabolism in humans and other species by 8%–56%, and can incur caloric costs of up to 2,000 kJ/day21 . The Immuno competence Handicap Hypothesis rose out of classical life-history theory and decades of research into costly signaling to become a dominant framework in the evolutionary ecology and behavioral endocrinology literature. It proposes that testosterone—as a critical mediator of male reproductive effort—suppresses immune function, and thus elaborate androgen based traits represent an honest signal of male quality since only high quality males can afford to incur such costs . While the ICHH is a dominant theoretical model in the life history literature, evidence in favor of this hypothesis is limited, and other lines of research have suggested alternative models to explain associations between androgens and immune function . The immuno suppressive effects of exogenously administered testosterone are well documented from experimental studies of nonhuman animal models . However,hydroponic containers recent studies and meta-analyses find mixed evidence that endogenous testosterone is an active immuno suppressant in free-living mammals .
In fact, naturalistic studies often show positive associations between endogenous testosterone and induced antibody response, suggesting that males in good condition can afford to maintain both high testosterone levels and robust immune responses . These findings are bolstered by experimental work in a reptile model showing that immune function is enhanced when exogenous testosterone is paired with food supplementation, but without food supplementation exogenous testosterone results in decreased innate immune function . Importantly, testosterone is down-regulated following infection and tissue injury, making it difficult to isolate the effects of testosterone on immune function in cross-sectional studies . While meta-analyses suggest that testosterone is overall immuno suppressive , there is still ambiguity depending on which aspects of immune function are studied, and whether the impacts of testosterone on immune function are direct or indirect. Another major issue is that most studies assessing trade-offs between immune function and testosterone rely on only a single biomarker of immune function, precluding the ability to examine a potential immuno modulatory role of testosterone. Immune function involves multiple coordinated responses, each with their own costs, benefits and interactions with other immune and endocrine responses. Numerous cytokines, for example, play critical roles in immune cell signaling and lymphocyte differentiation and have varying impacts on physiology and varying energetic costs . As an alternative to the ICHH, it has been proposed that testosterone is immuno-modulatory rather than immuno suppressive, that is, testosterone regulates trade-offs between different types of immune response . If only the energetically costlier forms of immunity are down regulated in higher testosterone males, then energetic availability may underlie some of the associations reported in the literature.
Indeed, evidence suggests that energetic costs can impact both testosterone levels and immune function . The ability to mount a rapid response to local infection or tissue injury is of particular utility, as high testosterone males more frequently engage in aggressive physical competition with other males . In mandrills, male–male competition in the form of physical aggression is a common route for spreading simian immunod eficiency virus , while physically aggressive interactions between wild rodent males increase transmission rates of hantavirus . Innate immune responses, which include pro- and anti-inflammatory cytokines, not only are crucial rapid responses to injury, but also play a role in subsequent wound healing . In free-ranging baboons, where success in male–male physical conflict determines males’ reproductive access to females, high status males—who have higher testosterone than subordinate males — heal faster than subordinates , perhaps because of positive phenotypic correlation. Thus, while there may be trade-offs between testosterone and some more energetically costly aspects of immune function , one would not expect that testosterone would down-regulate all aspects of immune function equally. Studying the role of testosterone in modulating immune function under naturalistic conditions is notoriously difficult in humans, where ethical concerns limit the use of various experimental protocols commonly used in nonhuman animal models. Observational studies demonstrate that testosterone decreases immediately following illness or injury , which is consistent with a life history framework which posits that energetic stress shunts caloric resources from investment in reproductive effort toward immune function.
While it is clear that any energetic stress, whether due to immune activation from infection or caloric restriction , results in decreased testosterone production, it is less clear that endogenous testosterone actively down-regulates human immune function. Indeed, a longitudinal study of Filipino males found a positive association between testosterone and immunoglobulin A , a marker of mucosal immunity .This study contributes to the literature with data from a free-living energy limited population living a relatively traditional lifestyle, use of multiple measures of immune activation, and measurement of biomarkers at baseline and following an ex vivo challenge. The analyses herein focus on 109 older adult men in a population of forager horticulturalists facing high pathogen burden . In this immunologically stressed population, we expect energetic trade-offs between testosterone and immune function to be stronger than that observed among energetically replete industrialized populations with lower infectious burden . Additionally, industrialized populations have significantly higher levels of testosterone at younger ages compared with subsistence populations, and steeper age-related declines . Most prior observational studies in humans have only measured circulating concentrations of immune markers under baseline conditions, as opposed to the immune response to challenge. Recent studies indicate that immune response to challenge is far costlier than baseline immune function, both in nonhuman animal models and humans . Given behavioral effects of androgens , individuals with higher testosterone may also engage in behavior that increases the likelihood of wounding or encountering pathogens . In this study, levels of urinary testosterone are examined in relation to 13 circulating cytokines following ex vivo whole blood antigen stimulation with a T-cell mitogen, phytohemagglutinin and a B-cell mitogen, lipopolysaccharides . PHA is a commonly used mitogen that activates the division and replication of T-cells, while LPS is a cell wall component of gram negative bacteria that binds to toll-like receptor 4 and initiates B-cell division and differentiation into plasma cells as well as activation of macrophages, monocytes, and dendritic cells . This study can therefore examine androgen mediated immuno-modulation in response to several common immune challenges facing humans. This experimental approach has major advantages; first, we are able to stimulate an immune response ex vivo which permits intra- and inter-individual comparisons in cytokine response to the same challenge controlling for baseline cytokine levels and other potential confounders. Additionally, while under normal physiological conditions circulating testosterone rapidly decreases following infection or tissue injury ,container raspberries using an ex vivo design we can examine the relationship between baseline testosterone and cytokine response to stimulation without any potential for steroidal down regulation post-infection. Given the advantages of this experimental design over previous observational studies, we hypothesize that we will have a level of contrast necessary to differentiate between broad immunosuppression versus immuno modulation of specific aspects of immune function. We hypothesize that higher endogenous testosterone will be associated with more down-regulation of energetically costly aspects of immune function, such as T-cell mediated immune responses, but not broad, generalized immunosuppression.Tsimane men came to the Tsimane Health and Life History Project’s clinic in San Borja, Beni, Bolivia to participate in THLHP protocols , and to receive a routine medical exam as part of the project’s behavioral–biomedical surveillance. All Tsimane aged 401 were invited to participate in this and other studies regardless of their health status, and approximately 85% of adults participated. Prior to the medical exam and interviews, men provided a first morning void urine specimen. Fasting blood was drawn, both with and without heparin as an anti-coagulant.
One vacutainer of blood without anticoagulant was allowed to clot, and then serum was separated via centrifugation and frozen in liquid nitrogen. Multiple 100 mL aliquots of heparinized whole blood were immediately added to separate round bottom microtiter wells in a sterile 96-well plate. The first aliquot received 100 mL of 20 mg/mL phytohaemagglutinin diluted in RPMI-1640, for a final concentration of 10 mg/mL PHA. The second aliquot received 100 mL of 20 mg/mL Lipopolysaccharides diluted in RPMI- 1640, for a final concentration of 10 mg/mL LPS. A third aliquot was mixed with 100 mL RPMI-1640 without mitogens. In all three cases, RPMI was supplemented with 100 IU/mL penicillin and 100 lg/mL streptomycin to prevent contamination. In the absence of a CO2 incubator in the San Borja clinic, the microtiter plate was sealed in a glass Tupperware with a lit candle, which burned the O2 in the container, thus enriching the CO2 concentration . The sealed plates were incubated at 378C for 72 hr. Following this, the supernatant was removed and frozen in liquid nitrogen for transport to UNM. Specimens were transported on dry ice and stored at 2808C for up to 2 years before assay.This study tested trade-offs among older Tsimane men between androgens and immune activation biomarkers by examining associations between endogenous testosterone and mitogen-stimulated cytokine levels. Our results indicate that endogenous testosterone is associated with immuno-modulation, or at least selectively suppressive as opposed to broadly immuno suppressive. Testosterone is associated with reduced cytokine responses following stimulation with a T-cell mitogen, PHA, while testosterone has no significant association with response to stimulation with a B-cell and monocyte mitogen, LPS. These results fit within a larger life-history and ecological immunology literature suggesting that men with higher levels of testosterone down regulate some, but not all aspects of immune activation. In this case we find an association suggesting that cytokine responses to T-cell mitogens are down regulated in higher testosterone males. Unlike B-cells, which can continue to produce relatively long lasting antibodies, cytotoxic T-cells must be continually produced and clonally expanded in large numbers. From an energetic perspective, T-cell mediated immune activation may be more costly than B-cell mediated immune activation due to this need to produce many cells . Thus, testosterone appears to selectively down regulate the most energetically expensive forms of immune activation, which is expected if testosterone serves an adaptive immuno-modulatory function, especially in energy-limited subsistence populations. Stimulation with PHA results in an immune response similar to that expected with exposure to viral infection , and tends to cause greater changes in IL-2, IL-4, IL-5, and IL-13 . In animal models, treatment with testosterone results in reduced response to viral infections, as measured by higher viral titers .Research on humans indicates that men are more susceptible to viral infections than women, and also that testosterone is down-regulated in men infected with diverse viruses ranging from influenza vaccinations to HIV . If men with higher testosterone have a decreased cytokine response to viral infection, then reducing levels of testosterone following infection would be an optimal response. Indeed, studies find decreases in testosterone following illness and tissue injury . Testosterone can be lowered by decreasing hypothalamicpituitary-gonadal production, or by increasing aromatization of testosterone to estrogen . Changes in testosterone and other biomarkers during and following illness may underlie some sickness behaviors like reduced physical activity and other depressive symptoms . Contrary to the results from PHA stimulation, cytokines produced by stimulation with LPS were not associated with testosterone. LPS stimulates B cells as well as macrophages, monocytes, and dendritic cells. Macrophages in particular may be important in response to injury, and so the maintenance of their activity with elevated testosterone might be important. However, B-cells are much more abundant in Tsimane blood compared with industrialized populations, while monocytes are very rare , so much of the LPS response is likely a B-cell response. Unlike T-cells that are lysed when destroying infections, B-cells that remain inactive are relatively low cost reservoirs that can be activated to produce antibodies as needed . While the developmental costs of producing B-cells and immunoglobulins can be high, the maintenance and activation costs are relatively low, as are the collateral costs in terms of tissue damage when activated . For a 10 kg human infant, the total cost of producing immunoglobulin G is approximately 0.043% of their daily protein budget, while the costs of B-cell proliferation during infection is estimated to be 0.00048% of this budget . Immunoglobulins have a half-life of approximately 25 days, and thus once produced, antibodies have protective effects that require little additional energetic maintenance or input .