Capturing systemic inflammation in a nonhuman primate using a new leukocyte activity assessment system

Abstract

We developed an optical system that simultaneously monitors leukocytes producing the reactive oxygen species (ROS) superoxide and hypochlorite ions using only a few microliters of whole blood. Here, we report the first use of this system to measure circulating blood from a nonhuman primate. This observational pilot study involved rhesus monkeys recruited for a different purpose, brain positron emission tomography in the conscious state. The results indicated that (1) surgical inflammation influences the total ROS produced by leukocytes per unit of time; (2) blood analysis of a septic rhesus monkey exhibited a characteristic pattern of leukocyte ROS production; and (3) a strong positive correlation was observed between leukocyte ROS production and neutrophil counts at normal C-reactive protein (CRP) levels, which decreased under abnormal CRP levels. These findings suggest that the system can capture systemic inflammation in non-human primates and highlight the importance of monitoring leukocyte ROS production to understand inflammatory status. The difficulty of identifying subjective symptoms in veterinary medicine highlights the importance of technologies for easily and objectively monitor animals’ physical conditions. Further analyses, including studies with larger animal populations and comparisons with conventional biomarkers, are needed to determine the specific inflammatory status reflected in leukocyte ROS production.

Introduction

Innate immunity is a fundamental defense mechanism in mammals,⁽¹⁾ with leukocytes serving as key indicators of physiological conditions, including resistance to pathogens and inflammatory status. Therefore, developing new technologies for evaluating leukocyte function will considerably advance veterinary medicine. While various automated technologies are available for measuring leukocyte counts and assessing their dynamics, including flow cytometry, which utilizes forward- and side-scattered light from blood cells,⁽²⁾ convenient methods for assessing leukocyte activity are limited despite their importance.^(3–5) Measuring leukocyte activity typically requires fractionation, such as through density gradient centrifugation,^(4,5) which involves blood sampling in milliliter volumes, a procedure invasive for small- and medium-sized mammals. Furthermore, fractionation requires technical expertise to minimize leukocyte damage and/or activation.

We have previously developed optical systems that can measure leukocyte activity using only 3 ‍μl of whole blood diluted into a reaction solution.^(6,7) Two types of measurement containers have been implemented: glass slides⁽⁶⁾ and specialized thin fluidic chips optimized for blood analysis.⁽⁷⁾ These systems are based on a technology that simultaneously measures both fluorescence (FL) and chemiluminescence (CL) from human cells.^(8–10) The developed system enables simultaneous monitoring of leukocytes that produce superoxide via chemiluminescence (CL-O₂^•−) and hypochlorite ions via fluorescence (FL-OCl⁻).^(6,7) O₂^•− is a primary metabolite of the respiratory burst of leukocytes, and OCl⁻ is a secondary metabolite catalyzed by myeloperoxidase (MPO) present in leukocyte granules.^(11,12) Additionally, simplified systems specifically designed for FL-OCl⁻ measurement have been developed.^(13,14)

Using our fluidic-chip-based system,⁽⁷⁾ we conducted several studies in humans, including analyses of daily variations in healthy volunteers,⁽⁷⁾ comparisons before and after treatment for peripheral arterial disease,⁽¹⁵⁾ effects of postprandial oxidative stress,⁽¹⁶⁾ and effects of adlay tea intake in healthy volunteers.⁽¹⁷⁾ Animal studies have also been conducted using a slide-glass-type system to analyze hemolyzed samples from rodent models, including mouse models of atherosclerosis⁽¹⁸⁾ and Alzheimer’s disease,⁽¹⁹⁾ and rat models of hypertension.⁽²⁰⁾ Nevertheless, to expand the applications of these systems in veterinary medicine, trials assessing blood samples from various non-rodent mammalian species are imperative.

Non-human primates, such as rhesus monkeys, are widely used in research models that are closely related to human biology and are important for both clinical and protective veterinary studies. Additionally, rhesus monkeys represent an excellent non-human primate model for studies on the aging process.^(21–24) Our institution, Central Research Laboratory of Hamamatsu Photonics K.K., houses rhesus monkeys for positron emission tomography (PET) research, providing convenient access to blood samples. Some monkeys were brought in for brain PET measurements in the conscious state. An acrylic head holder (4.5 ‍cm width × 5.5 ‍cm length × 3.5 ‍cm height) that enables the monkey’s head to be fixed noninvasively to a head holder on a modified monkey chair was used for the PET study.⁽²⁵⁾ Using the opportunity of the head holder mounting operation for PET measurement, it is possible to examine the effects of acute inflammation on the measurement values of the developed system. Therefore, we selected rhesus monkeys as a representative non-human primate species for this study. Monitoring the health status of medium-sized animals using only a few microliters of whole blood might be useful in veterinary medicine.

This study aimed to verify whether our system could effectively assess the inflammation of rhesus monkeys. An observational study that tracked daily variations in healthy human volunteers over several months suggested that FL-OCl⁻ may reflect individual physical states.⁽⁷⁾ We sought to determine whether a similar trend could be observed in rhesus monkeys and to investigate the factors influencing changes in CL-O₂^•− and FL-OCl⁻ levels. This observational pilot study utilized rhesus monkeys initially recruited for a different purpose, brain PET imaging in the conscious state. First, to investigate the effects of acute inflammation on measurement values, we compared data before and after mounting the head holder required for PET measurement in the conscious state. Additionally, we performed blood monitoring in a septic case and compared values between young male and old female monkeys. Finally, we performed correlation analyses between neutrophil count and CL-O₂^•− or FL-OCl⁻ values. Preliminary findings from this study were partially reported in Japanese literature,⁽²⁶⁾ and further analyses have now been completed and are presented here as original research.

Materials and Methods

Rhesus monkeys

Table 1 shows the profiles of the ten rhesus monkeys used in this study. A total of five young male (4–10-year-old, monkeys A–E) and five aged female (26–28-year-old, monkeys a–e) rhesus macaque monkeys (Macaca mulatta) obtained from Hamri Company, Ltd. (Koga, Japan) were included. The young male monkeys weighed 3.9–5.8 ‍kg, and the aged monkeys weighed 3.5–4.7 ‍kg. The monkeys were housed in NIH standard adapted stainless steel cages with controlled humidity (50 ± 20%) and temperature (24 ± 3°C) under a 14 ‍h light/10 ‍h dark cycle. They had auditory, visual, and olfactory contact with other monkeys. They were fed 120 ‍g of chow (Certified Primate Diet 5048; PMI Nutrition, St. Louis, MO) in the morning and were hand-fed various vegetables and fruits several days per week. To assess the health of the monkeys, their weights were routinely monitored, and expert veterinarian assistance was available on-site.

Table 1.

List of rhesus monkeys used in this study

Individual ID	Age	M/F	Category
A	4	M	Young
B	6	M	Young
C	5	M	Young
D	5	M	Young
E	10	M	Young (sepsis)
a	28	F	Old
b	28	F	Old
c	26	F	Old
d	28	F	Old
e	28	F	Old

Coincidentally, one young male monkey (10 years old, 5.8 ‍kg, monkey E) fell into critical condition. The monkey was deemed incapable of recovery and euthanized. According to the euthanasia report, the possible names of the disease were pyloric transit obstruction, aspiration pneumonia (concomitant bacterial infection), and septicemia. Supplemental Note* provides the details of the euthanasia report.

This study was approved by the Animal Care and Use Committee of Hamamatsu Photonics K.K. (approval no. HPK-2019-13A and -13B) and was carried out in accordance with the recommendations of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Surgical procedures

Experimentally naive monkeys were brought in for brain PET measurements in the conscious state. An acrylic head holder (4.5 ‍cm width × 5.5 ‍cm length × 3.5 ‍cm height) that enables the monkey’s head to be fixed noninvasively to a head holder on a modified monkey chair was used for the PET study.⁽²⁵⁾ A surgery was aseptically performed for the implantation of this head holder onto the skull. The monkeys were anesthetized with 1.5–2% isoflurane in nitrous oxide/oxygen (2/1) following initial sedation with ketamine-HCl (5 ‍mg/kg). After fixating the head to the stereotaxic frame, the skull was exposed by incision of the scalp with a sterile scalpel. Several small holes were carefully drilled into the skull. Polycarbonate anchor bolts were implanted through these holes and fixed with dental acrylic cement. An arch-shaped cap was then molded over these bolts with the appropriate amount of dental cement to restrict movement in the horizontal plane. During the recovery period after the operation (for at least 2 weeks), the monkeys were carefully treated with antibiotics and analgesics. The acrylic head holder was not removed from the monkeys’ skulls at the end of the experiment. The monkeys were not noticeably disturbed by the acrylic head holder because they were small and light. They moved freely in their cages and ate food without difficulty, as they did before surgery.

Young male monkeys A–D underwent surgery to mount a head holder for PET research during the study period. All old female monkeys (monkeys a–e) and young male septic monkey (monkey E) had undergone surgery in the past (head holders were mounted).

Blood collection

On blood sample day, except for surgery, the monkeys were deprived of food 12 ‍h before blood sampling. After fixing the monkey in the cage with a squeezing device, venous blood (200 ‍μl) was collected. Blood was stored at room temperature in a dipotassium ethylenediaminetetraacetic acid-coated BD Microtainer tube (Becton Dickinson and Company, Franklin Lakes, NJ) and used for measurements within 2 ‍h of collection. Blood samples at the end of surgery were taken under isoflurane anesthesia.

Measurement of CL-O₂^•− and FL-OCl⁻ by CFL-H2200

To monitor the leukocyte activity in rhesus monkey blood, the CFL-H2200 system was used for simultaneous measurement of CL-O₂^•− and FL-OCl⁻.⁽⁷⁾ The measurement procedure was the same as described in a previous report.⁽⁷⁾ Briefly, whole blood was diluted to 750 ‍μl of reaction solution kept at 37°C, containing 1 ‍mM CaCl₂, 0.5 ‍μM of the CL reagent 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one hydrochloride (MCLA; Tokyo Kasei, Tokyo, Japan), and 10 ‍μM of the fluorescent reagent aminophenyl fluorescein (APF; Goryo Chemical, Sapporo, Japan), and incubated at 37°C for 1 ‍min in the dedicated fluidic-chip. The fluidic chip was set in CFL-H2200, and CL-O₂^•− and FL-OCl⁻ were then measured simultaneously for 3,000 ‍s. The leukocyte stimulant, phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Tokyo, Japan), was added 150 ‍s after the start of measurements at a final concentration of 0.1 ‍μM. The measured data were analyzed using dedicated analysis software to calculate the total amount of CL-O₂^•− and FL-OCl⁻ signals and their rising points (RPs).

Measurement of neutrophil count and C-reactive protein (CRP)

Neutrophil counts and CRP levels in the blood were measured using Pentra MS CRP (Horiba, Kyoto, Japan). The instrument is for human blood measurements. The normal range of CRP was considered to be ≤0.3 ‍mg/dl, as in humans, according to the guidelines of the Japan Society of Clinical Chemistry.

Statistical analyses

Changes in leukocyte activity of four young male monkeys (A–D) before, immediately after, and one day after surgery were analyzed by parametric multiple pairwise comparisons using Dunnett’s test. For monkeys C and D, the most recent values before surgery were used (Pre 2 for monkey C and Pre 4 for monkey D). The Dunnett’s test was conducted using the SPSS ver. 26 software package (IBM, Tokyo, Japan). A comparison of leukocyte activity of four young male monkeys (A–D) before and approximately one month after surgery was analyzed using a paired t test. All values under normal conditions (CRP in the normal range) were used. For the values in monkeys C and D before surgery, the averages of all measured data were used. Approximately one month after surgery, the data from 30, 41, and 71 days after surgery were used for monkey A; 23, 33, and 35 days for monkey B; 28, 30, and 34 days for monkey C; 33 and 41 days for monkey D. The most recent values before surgery were used (Pre 2 for monkey C and Pre 4 for monkey D). The comparison of leukocyte activity between young male and old female monkeys was assessed using an unpaired t test. All used values were under normal conditions (CRP in the normal range). Both paired and unpaired t tests were conducted using Excel.

Results

Effects of surgery on young male rhesus monkeys

Blood samples were collected before and after surgery to mount the head holder, which was necessary for brain PET measurement in the conscious state. Figure 1 and Supplemental Table 1* show the results for young male monkey A. Figure 1A shows the changes in CL-O₂^•−, FL-OCl⁻, neutrophil counts, and CRP levels several days after surgery, and Supplemental Table 1* presents the results of all measurements. We also calculated CL-O₂^•− and FL-OCl⁻ per unit number of neutrophils (CL-O₂^•−/unit and FL-OCl⁻/unit, respectively) by dividing CL-O₂^•− and FL-OCl⁻ by the number of neutrophils. “Surgery” represents when the monkey is ready for blood collection immediately after the surgery; “anesthesia”, immediately after anesthesia; “cut”, immediately after scalpel insertion into the head; and “pre”, before surgery (on a different day than the day of surgery). The number of days since surgery is also indicated. Figure 1B shows the simultaneous measurement data for CL-O₂^•− and FL-OCl⁻. Immediately after anesthesia, leukocyte activity (CL-O₂^•−, CL-O₂^•−/unit, FL-OCl⁻, and FL-OCl⁻/unit) and neutrophil counts increased compared to those before surgery, which might reflect stress during anesthesia, such as intubation. Immediately after cutting, these values decreased to approximately the same levels as before surgery. Immediately after surgery, CL-O₂^•−, FL-OCl⁻, and neutrophil counts increased by approximately three times compared to those before surgery, while CRP was undetectable. These values increased further 1 day after surgery, and the CRP levels were above the normal range. These changes immediately after surgery might reflect the acute inflammation induced by mounting the head holder. CL-O₂^•−, FL-OCl⁻, and neutrophil counts decreased 2 days after surgery, and CRP levels returned to the normal range 3 days after surgery. However, on days 41 and 71 after surgery, the CL-O₂^•−, FL-OCl⁻, and neutrophil counts increased to levels close to those immediately after surgery, for unknown reasons. In contrast, CL-O₂^•−/unit and FL-OCl⁻/unit maintained relatively constant levels during this period.

Fig. 1.

Variations in leukocyte activity, neutrophil counts, and CRP levels in rhesus monkey A before and after surgery. (A) Scatter plot of them; (B) time course signal of CL-O₂^•− and FL-OCl⁻.

Figure 2 and Supplemental Table 2* show the results for young male monkey B, and Fig. 3 and Supplemental Table 3* show the results for young male monkey C. We measured the blood of young male monkey C before surgery on two different days, and the average values are shown in Fig. 3. The figure display is the same as described above for young male monkey A. Additionally, no blood sampling was performed during the surgery (“anesthesia” and “cut”). In young male monkeys B and C, similar to young male monkey A, CL-O₂^•−, FL-OCl⁻, and neutrophil counts increased approximately 2–3 times immediately after surgery compared to before surgery. These values decreased over time, suggesting that the acute inflammatory state had recovered. The CRP levels of these monkeys peaked 1 day after surgery, later than the CL-O₂^•−, FL-OCl⁻, and neutrophil counts, and returned to the normal range approximately 1 month after surgery. The CL-O₂^•−/unit and FL-OCl⁻/unit levels in these monkeys remained relatively constant throughout this period. However, the CL-O₂^•−/unit levels tended to increase from 1 to 2 days after surgery.

Fig. 2.

Variations in leukocyte activity, neutrophil counts, and CRP levels in rhesus monkey B before and after surgery. (A) Scatter plot of them; (B) time course signal of CL-O₂^•− and FL-OCl⁻.

Fig. 3.

Variations in leukocyte activity, neutrophil counts, and CRP levels in rhesus monkey C before and after surgery. (A) Scatter plot of them; (B) time course signal of CL-O₂^•− and FL-OCl⁻.

Figure 4 and Supplemental Table 4* show the results for young male monkey D. We measured the blood of young male monkey D before surgery on four different days, and the average values are shown in Fig. 4. In young male monkey D, the increases in CL-O₂^•−, FL-OCl⁻, and neutrophil counts immediately after surgery were smaller than those in young male monkeys A–C (within 1.5 times compared to before surgery levels); however, these values were increased to approximately three times 1 day after surgery compared to those before surgery. The CRP level of monkey D on 1 day after surgery was 4.1 ‍mg/dl, which was higher than those of monkeys A–C. Furthermore, monkey D still had relatively high CL-O₂^•−, FL-OCl⁻, and neutrophil counts (approximately 2.5–3.0 times higher than that before surgery) and CRP (1.1 ‍mg/dl) at 22 days after surgery. Subsequently, the values gradually decreased. CL-O₂^•−/unit and FL-OCl⁻/unit remained at relatively constant levels throughout this period.

Fig. 4.

Variations in leukocyte activity, neutrophil counts, and CRP levels in rhesus monkey D before and after surgery. (A) Scatter plot of them; (B) time course signal of CL-O₂^•− and FL-OCl⁻.

Figure 5A and B show the changes in leukocyte activity in four young male monkeys (A–D) before surgery, immediately after surgery, and 1 day after surgery. In addition, Supplemental Fig. 1A* shows a comparison of the time from the addition of the stimulant to the increase in CL-O₂^•− and FL-OCl⁻ signals (rising point, RP).⁽¹⁵⁾ The statistical analysis (Dunnett’s test) showed that, compared to before-surgery levels, CL-O₂^•− was significantly increased immediately after surgery (p<0.05) and 1 day after surgery (p<0.01), and FL-OCl⁻ was significantly increased 1 day after surgery (p<0.05) (Fig. 5A). However, no statistically significant difference was observed in CL-O₂^•−/unit, FL-OCl⁻/unit, CL-O₂^•− RP, and FL-OCl⁻ RP levels in a similar comparison (Fig. 5B and Supplemental Fig. 1A*). Interestingly, CL-O₂^•−/unit tended to increase with time from surgery (Fig. 5B).

Fig. 5.

Bar graphs comparing leukocyte activity between before and after surgery in young male monkeys (A–D, n = 4). (A) Comparison of CL-O₂^•− or FL-OCl⁻ between before surgery (Pre), immediately after surgery (Surgery), and 1 day after surgery (1 day); (B) comparison of CL-O₂^•−/unit or FL-OCl⁻/unit. The data of (A) and (B) were analyzed by parametric multiple pairwise comparisons using Dunnett’s test. (C) Comparison of CL-O₂^•− or FL-OCl⁻ between before surgery (Pre) and approximately 1 month after surgery (1M); (D) comparison of CL-O₂^•−/unit or FL-OCl⁻/unit. The data of (C) and (D) were analyzed using a paired t test. *p<0.05, **p<0.01.

Figure 5C, D, and Supplemental Fig. 1B* show the comparison of leukocyte activity in four young male monkeys (A–D) before and approximately 1 month after surgery. The statistical analysis (paired t test) showed that CL-O₂^•− (p<0.05) and CL-O₂^•−/unit (p<0.05) significantly increased approximately 1 month after surgery (Fig. 5C and D), suggesting a continued inflammatory status. However, no statistically significant difference was observed in FL-OCl⁻, FL-OCl⁻/unit, CL-O₂^•− RP, and FL-OCl⁻ RP levels in a similar comparison (Fig. 5C, D, and Supplemental Fig. 1B*).

Comparisons among individual young male monkeys are difficult because of fluctuations due to surgery; however, when comparing before-surgery values, CL-O₂^•− and CL-O₂^•−/unit tended to show high values in monkeys C and D. Particularly, monkey D had a high CL-O₂^•−/unit value. However, monkeys A–D had similar values of FL-OCl⁻ and FL-OCl⁻/unit. In addition, the time-course signal of CL-O₂^•− presented several patterns. Monkeys A, B, and D displayed bimodal peaks, while monkey C showed a unimodal peak. The initial peak for monkey D was higher than that of monkeys A and B.

Blood measurement of old female rhesus monkeys

Supplemental Table 5* shows the results for the old female rhesus monkeys (n = 5). Two measurements were taken on separate days for each monkey. Figure 6 shows the representative measurements (Measurement 1) for the old female monkeys. For each measurement value, individuals were ranked, although there was some variation between the two measurements. For FL-OCl⁻ and FL-OCl⁻/unit, the order was the monkey d > c > b > a > e (d: high; c and b: moderate; a and e: low). A similar tendency was observed for neutrophil counts. In contrast, for CL-O₂^•−, the order was d > c > e > b > a (d: high; c and e: moderate; b and a: low), and for CL-O₂^•−/unit, e > c > d > b > a (e: high; c and d: moderate; b and a: low). Monkey e was characterized by a higher CL-O₂^•−/unit value than the other monkeys. In monkey d, the FL-OCl⁻, FL-OCl⁻/unit, and neutrophil counts were higher than those in the other old female monkeys, and the CRP values were abnormal, suggesting an inflammatory disorder. In addition, the time-course signal of CL-O₂^•− showed a bimodal peak in monkeys a–c, whereas monkeys d and e showed unimodal peaks. Monkeys d and e appeared to have a unimodal peak, but the second peak might be hidden by the higher first peak.

Fig. 6.

Time course signal of CL-O₂^•− and FL-OCl⁻ in old female rhesus monkeys.

Comparison between young male and old female rhesus monkeys

Figure 7A, B, and Supplemental Fig. 2A* show the comparison of leukocyte activity in four young male monkeys (A–D) before surgery and in old female monkeys; Figure 7C, D, and Supplemental Fig. 2B* show the comparison of leukocyte activity in four young male monkeys (A–D) approximately 1 month after surgery and in old female monkeys. All old female monkeys had previously undergone surgery to mount their head holders. Data from days with abnormal CRP levels were excluded from the comparison. No statistically significant differences were observed between young male and old female monkeys (unpaired t test).

Fig. 7.

Bar graphs comparing leukocyte activity between young male (A–D, n = 4) and old female rhesus monkeys (a–c and e, n = 4). The data were analyzed by an unpaired t test. (A) Comparison of CL-O₂^•− or FL-OCl⁻ between young male monkeys before surgery (young male Pre) and old female monkeys (old female); (B) comparison of CL-O₂^•−/unit or FL-OCl⁻/unit. (C) Comparison of CL-O₂^•− or FL-OCl⁻ between young male monkeys approximately 1 month after surgery (young male 1M) and old female monkeys (old female); (D) comparison of CL-O₂^•−/unit or FL-OCl⁻/unit.

Blood measurement of a sepsis rhesus monkey

Figure 8 and Supplemental Table 6* show the results for monkey E scheduled for euthanasia, owing to poor physical condition. As monkey E underwent surgery to mount the head holder, data from young male monkeys obtained approximately 1 month after the surgery are shown for comparison. After this measurement, monkey E was euthanized, and an autopsy was performed by a veterinarian. The euthanasia report showed that the possible names of the disease were pyloric transit obstruction, aspiration pneumonia, and septicemia (see also Supplemental Note*). The infection was thought to have occurred approximately 1 month before the blood measurement. For simplicity, we refer to this case as a sepsis monkey in this study. The blood was collected immediately before euthanasia. The neutrophil count in monkey E was considerably lower (0.67 × 10³ cells/μl) than the average of other monkeys, which may reflect the consumption of circulating neutrophils by the infection. Compared with that in young male monkeys, CL-O₂^•− in monkey E was approximately 1/5, whereas FL-OCl⁻ was approximately 2.5 times higher. Furthermore, in monkey E, the CL-O₂^•−/unit was similar to or slightly higher than those in young male monkeys, whereas the FL-OCl⁻/unit was over 10 times higher. A similar tendency was observed when monkey E was compared with young male monkeys before surgery and with old female monkeys. Moreover, the RPs of CL-O₂^•− and FL-OCl⁻ were longer in monkey E (CL-O₂^•− RP: 172 ‍s; FL-OCl⁻ RP: 382 ‍s) than in young male monkeys (Fig. 8 and Supplemental Table 6*), suggesting that leukocyte reactivity was blunted owing to continuous severe inflammation.

Fig. 8.

Time course signal of CL-O₂^•− and FL-OCl⁻ for comparison in sepsis and young male rhesus monkeys.

Correlation between neutrophil count and CL-O₂^•− or FL-OCl⁻

Figure 9A and B show the results of the correlation analyses of neutrophil count and CL-O₂^•− or FL-OCl⁻ for all collected data (n = 46); Figure 9C and D, for only normal conditions (CRP within the normal range, n = 36); Figure 9E and F, for only abnormal conditions (CRP outside the normal range, n = 10). The p values for the correlation analyses are shown in each graph. The neutrophil count and CL-O₂^•− demonstrated a high positive correlation (correlation coefficient, R, was approximately 0.8) in all cases. In contrast, although the correlation between neutrophil count and FL-OCl⁻ showed a strong positive correlation under normal CRP levels, it decreased under abnormal CRP levels.

Fig. 9.

Scatter plots showing the correlation between neutrophil counts and CL-O₂^•− or FL-OCl⁻ levels. (A) and (B) show the results for all obtained data (n = 46); (C) and (D), that for only normal conditions (CRP within normal range, n = 36); (E) and (F), that for only abnormal conditions (CRP outside normal range, n = 10).

Discussion

In this study, blood samples from the rhesus monkeys were analyzed using the developed system CFL-H2200 to monitor leukocyte activity. Our results suggest that CFL-H2200 can capture changes in the systemic inflammatory status of non-human primates.

In young male monkeys A–D, CL-O₂^•−, FL-OCl⁻, and neutrophil counts tended to increase immediately after surgery compared with those before surgery, even though the inflammatory marker CRP was in the normal range (Fig. 1A, 2A, 3A, 4A, and Supplemental Table 1–4*). These results are reasonable because leukocyte activity, CL-O₂^•−, FL-OCl⁻, and neutrophil counts are the causes of inflammation, and CRP reflects the results. These features were also observed in a previous human study.⁽⁷⁾ Statistical analyses also suggest that acute inflammation associated with surgery increases leukocyte-produced reactive oxygen species (ROS), CL-O₂^•− and FL-OCl⁻ levels (Fig. 5A). Particularly, CL-O₂^•− was increased immediately after surgery, earlier than the development of CRP (Fig. 1A, 2A, 3A, 4A, 5A, and Supplemental Table 1–4*). Furthermore, in young male monkeys A–D, each value was lower approximately 1 month after surgery than immediately after surgery, but was higher (1.5–2 times) than that before surgery (Fig. 1A, 2A, 3A, 4A, 5A, and Supplemental Table 1–4*). Statistical analyses also revealed that CL-O₂^•− and CL-O₂^•−/unit levels were higher approximately 1 month after surgery than those before surgery (Fig. 5C and D), suggesting that the inflammatory status continues even approximately 1 month after surgery.

There is no statistical difference in leukocyte activity between young male and old female rhesus monkeys (Fig. 7 and Supplemental Fig. 2*). Nonetheless, the mean values of CL-O₂^•−/unit and FL-OCl⁻/unit tended to be higher in old female monkeys than those in young male monkeys (Fig. 7B and D), possibly reflecting differences in leukocyte quality. However, since the monkeys were not recruited exclusively for this observational pilot study, standardizing their characteristics was limited. All young monkeys were male, and all old monkeys were female (Table 1). Consequently, we were unable to distinguish the effects of monkey age and sex on leukocyte activity. This is an issue that needs to be addressed in future research.

In sepsis, a characteristic pattern of leukocytes producing ROS has been suggested: low CL-O₂^•− value is associated with low neutrophil counts, whereas high values are observed in FL-OCl⁻ and FL-OCl⁻/unit (Fig. 8 and Supplemental Table 6*). Neutrophil extracellular traps (NETs) are involved in sepsis complications, including thrombosis, and MPO, which catalyzes OCl⁻ production, is involved in NET formation.⁽²⁷⁾ The characteristic pattern of leukocytes producing ROS might be associated with NET formation.

Correlation analysis of leukocyte counts with leukocyte activity revealed that FL-OCl⁻ showed a strong positive correlation under normal CRP levels, it decreased under abnormal CRP levels (Fig. 9), suggesting that the neutrophil count and FL-OCl⁻ levels reflect different biological information, particularly during inflammation. Further analyses are important to determine what kind of information on the physical condition FL-OCl⁻ reflects, including increasing the number of animals and comparing it with conventional biomarkers.

The blood composition of rhesus monkeys is similar to that of humans,^(28,29) and neutrophil MPO is present in the blood.⁽³⁰⁾ Our results demonstrated that neutrophil counts in rhesus monkeys were comparable to those in humans. In this study, CL-O₂^•− and FL-OCl⁻ were detected upon PMA stimulation, suggesting that O₂^•− production by NADPH oxidase and the subsequent OCl⁻ production catalyzed by MPO occur in rhesus monkeys as well as in humans. CL-O₂^•− and FL-OCl⁻ levels are similar in both species.⁽⁷⁾ We consider leukocytes, mainly neutrophils, to be the FL-OCl⁻ and CL-O₂^•− sources, as observed in humans.^(6,7) However, identifying these sources, such as through measurements of isolated neutrophils and the use of neutrophil activity inhibitors, as demonstrated in human blood,^(6,7) remains an important future task.

In some rhesus monkeys, the time-course signal of CL-O₂^•− clearly demonstrated bimodal peaks (Fig. 1B, 2B, 4B, and 6), which have not been observed in human blood measurements to date.^(7,13,15,16) It is possible that the two peaks were not separated in human blood measurements; however, in rhesus monkeys they tended to be distinct. The signaling pathway that induces O₂^•− production in rhesus monkeys may differ from that in humans. To clarify this, validation using stimulants other than PMA and inhibitors of store-operated calcium entry (SOCE)⁽³¹⁾ is pertinent.

Author Contributions

KT concepted this study, analyzed and interpreted the data, and wrote the original draft. SY and DF conducted animal experiments, interpreted the data, and wrote the original draft. YH conducted the blood measurements and analyzed the data. TK conducted animal experiments. YS analyzed and interpreted the data. KK designed and concepted this study, analyzed and interpreted the data, and wrote the original draft. All the authors contributed to and approved the final version of the manuscript.

Conflict of Interest

KT, SY, DF, TK, YS, and KK are employees of Hamamatsu Photonics K.K. YH was an employee of Hamamatsu Photonics K.K. during the time this research was conducted. KT, YH, and KK have submitted patent applications for the technologies related to this study. The funders had no role in the data analysis and interpretation.