Differential Gender Effects of a Reduced Calorie Diet on Systemic Inflammatory and Immune Parameters in Nonhuman Primates

Abstract

Dietary manipulation, including caloric restriction, has been shown to significantly impact host response capabilities, particularly associated with aging. This investigation compared systemic inflammatory and immune response molecules in rhesus monkeys (Macaca mulatta) on continuous long term calorie-restricted (CR) diets with a matched group of animals on a control diet, examining the effects of both gender and aging. The results demonstrated that haptoglobin and α₁anti-glycoprotein were elevated in serum of male monkeys. Serum IgG antibody responses to C. rectus, A. actinomycetemcomitans, and P. gingivalis were significantly elevated in female monkeys. While only the antibody to F. nucleatum was significantly affected by the calorie-restricted diet in females, antibody levels to P. intermedia, C. rectus and T. denticola demonstrated a similar trend. In this investigation, only selected serum antibody levels were influenced by the age in male animals, seemingly related to increasing clinical disease in this gender. More generally, analytes were modulated by gender and/or diet in this oral model system of mucosal microbial challenge.

Introduction

The inflammatory response involves humoral and cellular responses to a given challenge. There has been an increasing demand to assess the effects of aging on immune cell functions. It has been well documented that T cell and B cell numbers and functions decrease progressively during aging (1), although the impact of aging on innate immunity remains to be clarified. Numerous studies in rodent models have documented a decline in immune responsiveness with age (2–5). In particular, these studies have indicated that advancing age produces a general depression in the adaptive immune response (5), accompanied by an increase in the production and release of reactive oxygen species, reactive nitrogen species, and the activity of cyclooxygenase enzymes with an accompanying increase in prostaglandin production (1, 3, 4). In addition, there appears to be an up-regulation of inflammatory cytokine gene expression with aging including TNFα, IL-1, IL-6, INFγ, and TGFβ [(5–7). However, assessing the impact of aging on cellular functions in humans is complicated by the effects of chronic diseases frequently observed in elderly persons. Thus, in human systems it continues to be a challenge to delineate the effects of aging versus the effects of systemic or environmental conditions (8).

Caloric restriction (CR) of dietary intake has been shown to significantly alter a wide range of biological processes and, in particular, attenuate age-related disease in rodent models of aging (4, 8–11). This dietary manipulation has been demonstrated to attenuate the development of oxygen radical induced cell damage, to maintain more robust host responses protecting against deleterious extrinsic and intrinsic challenges to normal cell, tissue, and organ function, and to maintain general body-wide physiologic functions (12–25). Recent studies have interpreted these macro-observations at the molecular level by identifying that CR could stop aging-associated changes in the expression of numerous genes (12, 13), including altering insulin-like growth factor 1 (IGF-1) associated with age-related decreases in insulin sensitivity (20, 26, 27). Only recently have reports emerged regarding the potential for this dietary manipulation to also alter physiologic parameters in nonhuman primates, a species more closely related to humans (28–36). Since many of these findings are similar to those seen in rodent models, the nonhuman primates may provide a valuable link between rodent studies of reduced calorie diets and application of this approach to a human population.

Periodontal disease is a predominant chronic inflammatory disease of mankind (37–39) that is a consequence of oral infection, chronic inflammation, and destruction of collagen and bone, and can be documented to occur naturally with aging in humans and nonhuman primates (37, 40, 41). The extent and severity of tissue destruction is affected by the magnitude and characteristics of the host response and may be modulated by environmental, systemic or genetic factors (38, 39, 42). Periodontal destruction is cumulative and not naturally reversible, thus, it is unclear as to whether aging impacts the rate of disease progression or just reflects the accumulation of disease over time (41, 43). The importance of periodontal disease as a model of host-bacterial interactions, inflammation, and inflammatory disease lies in the ability to isolate and characterize bacterial and host factors from the oral cavity in a non-invasive manner and to correlate these changes with host tissue pathology. The nonhuman primate model has provided the essential bridge for understanding the interaction of the subgingival microbiota with the inflammatory/immune response targeted to selected members of this microbiota (44–48). Increasing evidence also suggests that these oral microorganisms can translocate to the systemic circulation and may routinely stimulate the reticuloendothelial and immune systems (49–51). Recent studies have provided clear evidence that the oral cavity can function as a nidus for a variety of potential medical problems (49, 51, 52). Several members of the periodontopathic microbiota have been found to be involved in other systemic infections, as well as in the induction of an acute phase response (APR) (53). Increased levels of acute phase proteins have been identified in adult patients with periodontitis, e.g. CRP, haptoglobin, and may reflect the infection and manifestations of acute and chronic inflammation that exist in the periodontium (53–56). Moreover, it was also evident that patients exhibiting the most severe disease had the greatest levels of each of the acute phase reactants. In addition, a serum antibody response is observed in these localized periodontal infections. It has been suggested that this serum response may reflect a local gingival inflammatory/immune response to the bacteria. Thus, the systemic antibody response observed in periodontitis patients appears to result from specific elicitation of antibody to an infection with the microorganism (50, 57, 58).

This study utilizes the accessibility and natural development of chronic inflammation and disease in the oral cavity to examine the effects of long term dietary calorie restriction on inflammatory/immune responses in a human-like model system, the rhesus monkey.

Materials and methods

Animals and diet

Eighty-three rhesus monkeys (Macaca mulatta) that are part of an ongoing study of caloric restriction and aging were used in these studies (Table 1). These animals have been housed at the National Institutes of Health Animal Center, Poolesville, MD. Details of the study have been described previously (Reynolds M, G. Branch-Mays, D. Dawson III, K.F. Novak, J. Mattison, J. Gunsolley, D. Ingram, M. Lane, G. Roth, and M.J. Novak. Effects of dietary calorie restriction on inflammatory disease in a nonhuman primate model. Submitted). At the time of the current study, animals had been assigned to continuous long term CR or control diets for periods of 13–17 years.

Table 1.

Age distribution of nonhuman primate cohort in study.

Gender	Diet Group	N	Mean ± SD
Female	CON	19	18.74 ± 1.29
	CR	16	16.94 ± 1.22
Male	CON	26	22.35 ± 1.21
	CR	20	22.70 ± 1.53

Serum analyses

Blood was collected from all monkeys under ketamine or telazol anesthesia following an overnight fast, serum was separated and stored at −80°C until assay, when IgG antibody to six oral bacteria was evaluated using an ELISA as described previously (59, 60). Briefly, Campylobacter rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Treponema denticola, and Porphyromonas gingivalis were grown in broth under anaerobic conditions, harvested by centrifugation, formalin-killed, washed, and stored at −20°C for use as antigens (61).

Selected acute phase reactants were quantified using ELISA procedures developed in our laboratory (53, 56, 62). Specifically we examined levels of C-reactive protein (CRP), haptoglobin (HG), fibrinogen (Fib), α₁-antiproteinase (α₁-AT), and α₁-acid glycoprotein (α₁-AG) in serum samples from all animals.

Statistical analyses

In the primary analysis, the effects of age and CR were analyzed separately by gender due to different age distributions (Table 1). Age was modeled as a linear variable. In secondary analyses, data were submitted to a linear regression analysis in which gender was included in the model. The purpose of the secondary analysis was to verify the robustness of the results. Statistical analysis was performed using JMP (SAS, Inc.). Statistical significance was set at an alpha level of 0.05.

Results

Systemic acute phase reactants

The levels of various acute phase reactants were determined in serum from each monkey and compared based upon gender and diet. Fig. 1 demonstrates that haptoglobin, and α1-antiglycoprotein were significantly greater in males compared to females and were not affected by CR diet.

Figure 1.

Acute phase reactants in serum from nonhuman primates categorized based upon gender (F – female; M – male) and diet (CR – calorie restricted; CON – control ad libitum). The bars denote the mean levels of each mediator (HG – haptoglobin; FIB - fibrinogen; CRP – C-reactive protein; α1-AT –α1-antiproteinase; α1-AG –α1-acid glycoprotein) and the vertical brackets denote 1 standard error.

Systemic antibody responses to oral bacteria

Figures 2–5 show the levels of serum IgG antibody to a group of oral bacteria commonly associated with periodontal disease (63, 64). In Fig. 2 antibody levels to A. actinomycetemcomitans and P. gingivalis were significantly elevated in the female monkeys compared to males with no effect of diet or age. In Fig. 3 the antibody to P. intermedia was significantly related to age in male animals, although the females did exhibit a trend toward higher levels of antibody, irrespective of diet. Fig. 4 illustrates that serum IgG antibody to F. nucleatum was significantly elevated by a CR diet in the females only, and the levels increased significantly with age in the males unrelated to diet. In Fig. 5 the serum antibody levels to C. rectus were also significantly elevated in females compared to males, and these levels increased significantly in males with age unrelated to diet.

Figure 2.

Serum IgG antibody levels to A. actinomycetemcomitans (Aa), P. gingivalis (Pg), and T. denticola(Td) in nonhuman primates categorized based upon gender and diet. The bars denote the mean levels of each mediator and the vertical brackets denote 1 standard error.

Figure 5.

Serum IgG antibody levels to C. rectus (Cr) in nonhuman primate categorized based upon gender and diet (LEFT). The bars denote the mean levels of each mediator and the vertical brackets denote 1 standard error. (RIGHT) Antibody levels related to age classified according to gender (green – female; blue – male). The dashed lines denote 95% confidence interval.

Figure 3.

Serum IgG antibody levels to P. intermedia (Pi) in nonhuman primates categorized based upon gender and diet (LEFT). The bars denote the mean levels of each mediator and the vertical brackets denote 1 standard error. (RIGHT) Antibody levels related to age classified according to diet (green –control; blue – calorie restricted). Dashed lines denote 95% confidence interval.

Figure 4.

Serum IgG antibody levels to F. nucleatum (Fn) in nonhuman primate categorized according to gender and diet (LEFT). The bars denote the mean levels of each mediator and the vertical brackets denote 1 standard error. (RIGHT) Antibody levels related to age classified according to gender (green – female; blue – male). The dashed lines denote 95% confidence interval.

Discussion

This investigation described the characteristics of systemic inflammatory and immune responses of a nonhuman primate cohort related to a calorie-restricted diet. This dietary manipulation has been demonstrated to contribute toward potential therapeutic outcomes related to biologic processes adversely affected by aging (10, 11, 15, 16). CR has been shown to minimize decline in specific immune functions (1, 5), as well as attenuate destructive inflammatory responses (4). Various physiologic parameters (4, 8, 14, 25, 35, 65) and hormonal changes (thyroid hormones, melatonin, and dehydroepiandrosterone sulfate) (19, 35, 66, 67) related to improved aging have been reported in nonhuman primates on a long-term CR diet.

In the current study, the nonhuman primate model was used to examine the effect of a calorie restricted diet on systemic inflammatory and antibody responses to oral commensal and opportunistic bacterial pathogens. Periodontal disease is a complex microbial infection with similarities between humans and nonhman primate (48, 54). This oral infection elicits a chronic immunoinflammatory lesion that destroys soft and hard tissues resulting in destruction of the periodontium (37, 68–70). While the extent and severity of periodontal disease is related to aging (41, 71), it is unclear if this finding represents a cumulative expression of years/decades of challenge to the tissues or an exacerbated disease process reflecting altered aging processes measured at a molecular level. Periodontal disease provides a model of host-bacterial interactions, inflammation, and adaptive immune responses that can be used to examine nutritional and aging changes in the oral cavity. In addition, ample evidence has demonstrated that these local oral infections also stimulate a systemic inflammatory and humoral immune response (50, 53, 58, 72–77).

We have previously reported an age-associated increase in periodontal disease in nonhuman primates (Reynolds M, G. Branch-Mays, D. Dawson III, K.F. Novak, J. Mattison, J. Gunsolley, D. Ingram, M. Lane, G. Roth, and M.J. Novak. Effects of dietary calorie restriction on inflammatory disease in a nonhuman primate model. Submitted). Periodontal disease was more prevalent in the males with a more dramatic effect related to aging. The current study suggested that characteristics of the systemic host responses were consistent with these disease findings. Systemic inflammatory mediators were significantly greater in male nonhuman primate compared to females. Human studies have shown that increased severity/extent of periodontitis results in higher serum levels of these host response molecules (53, 72, 78, 79). Thus, it was expected that the males would have elevated levels of these mediators. Although the male monkeys on a long term CR diet had generally lower levels across the profile of acute phase reactants, this difference was not statistically significant. These outcomes are consistent with cross-sectional and longitudinal observations of human populations demonstrating elevated acute phase reactants in periodontitis and a decrease in these mediators after mechanical and anti-inflammatory therapies (53, 72, 78, 79). Since these systemic inflammatory responses have been suggested to reflect and/or contribute to chronic inflammatory diseases, eg. cardiovascular and diabetes, the contribution of chronic periodontitis to these systemic biomolecules has been suggested to be a biologic link between oral and systemic diseases (80).

In humans, both the specificity and levels of serum antibody responses to oral pathogens are clearly related to periodontal disease (50, 58, 77, 81, 82). With increasing disease both antibody frequency and level also increase and various studies have demonstrated that these serum antibody levels will be elevated following mechanical therapy and will correlate with response to treatment (50, 83–86). Moreover, changes in serum antibody to selected oral pathogens appear to occur following emergence of the microorganisms in oral biofilm samples and prior to identification of progressing disease (50, 87). These findings suggest that the humoral immune response in local tissues, and reflected in the systemic circulation, is likely an important component of the host’s responses trying to re-establish homeostasis by controlling the challenge of these extracellular bacterial pathogens. Interestingly, we observed significantly elevated antibody to these oral pathogens in female monkeys who displayed less periodontal inflammation and disease than the males (Reynolds M, G. Branch-Mays, D. Dawson III, K.F. Novak, J. Mattison, J. Gunsolley, D. Ingram, M. Lane, G. Roth, and M.J. Novak. Effects of dietary calorie restriction on inflammatory disease in a nonhuman primate model. Submitted). The antibody responses also appeared to be generally elevated with CR, with the most substantive impact in females. These results suggested a gender specific differentiation of responses oriented towards a potentially destructive inflammatory response in males versus a protective adaptive immune response in females. This type of observation has a basis in existing data demonstrating inherent gender-based variations in levels of immunoglobulins (88–90) and other host response biomarkers (91, 92). Subsequent studies implementing a longitudinal, prospective design creating a ligature-induced periodontal challenge in these animals should help clarify the dynamics of the relationship of periodontal disease to these response profiles. Lastly, of the analytes measured, serum antibody levels demonstrated some positive correlations with aging, primarily in the males, consistent with increased clinical parameters of periodontal disease in this group.

These cross-sectional observations provide a snapshot of host serum acute phase and antibody responses in nonhuman primate. The response profiles supported an inherently different response pattern in monkeys that was gender determined, as well as differences in the genders with respect to the impact of CR on these systemic responses. Further analyses will determine the interaction of oral clinical presentation and these responses demonstrating the usefulness of the oral cavity as a model for aging studies of host-bacterial interactions.