A Whole Blood Method for Assessing the Innate Immune Response in Chickens

: Innate immunity is considered the first line of immune defense and is typically an unmea-sured response. Here we report a method for evaluating the innate immune response in chickens by using whole blood which has been activated by lipopolysaccharide (LPS) to induce IL-6 release by innate immune cells. It was found that a 24-h LPS activation time interval was the optimum time interval for inducing the IL-6 response. An activation index, defined as the PBS activated control response subtracted from the LPS activated response and then divided by the PBS activated control response and expressed as a percentage, was useful for demonstrating and comparing the magnitude of the innate immune response. Results indicated that there was wide variation between the IL-6 response between individual birds although statistically significant results were obtained for all individual birds at the 24-h activation time interval. The activation indices from all birds were greatest at the 24-h activation time interval. Statistically significant results were achieved when all the data from all birds at the 24-h activation time interval were combined. The cells responsible for the IL-6 response were identified as the peripheral blood monocytes.


Introduction
Immunity can be enhanced by using vaccines and adjuvants to stimulate the adaptive component of the immune response leading to increased humoral (i.e., antibody) and/or cell mediated responses.Using serologic assays to measure antibody levels is a useful, convenient, and cost-effective method to evaluate the humoral immune response.An equally important component of the immune system is the often unnoticed, and frequently unmeasured, innate immune response.Innate immunity is characteristically nonspecific, rapid, and the first line of immune defense.It is complementary to, and also paramount in initiating, the adaptive response [1].Boosting the innate immune response is a phenomenon referred to as trained immunity and has been demonstrated in vitro with chicken cells [2].There are several immune cells that contribute to innate immunity which include leukocytes such as neutrophils (heterophils in avian species), monocytes, macrophages, thrombocytes, and natural killer cells.These cells are involved in phagocytosis, antigen presentation as well as free radical and cytokine production [1].Interleukin-6 (IL-6) is a pro-inflammatory cytokine in chickens that is produced soon after the induction of the innate immune response [3].There are various methods for measuring the innate immune response, many of which are very technical, expensive, and labor intensive.Such methods may include separating cells and using cell surface markers and subjecting the cells to a fluorescentactivated cell sorting (FACS) technique using a flow cytometer [2,4], using upregulation of mRNA [4], and/or evaluating the amount of super oxide anion released as a result of inducing an oxidative burst [2,5].Here we report a method for evaluating the innate

Materials and Methods
Ethical statement.This study was conducted at the University of Nebraska-Lincoln.The protocols were approved by the Institutional Animal Care and Use Committee (Project ID 2114 and 1874).
Birds and blood samples.Adult (24 weeks of age) male and female specific pathogenfree (SPF) layer-type chickens were hatched (SPF eggs obtained from VALO BioMedia, Adel, IA,) and reared in a biologic containment level 2 facility.Birds used in this study were hatch mates.Three to five mL of blood was collected from the wing vein using a 25 ga needle and a 5 mL syringe containing 1000 units of sodium heparin.Following collection, the heparinized blood was kept at room temperature and used within 30 min of collection.
Media and reagents.Ross Park Memorial Institute (RPMI) 1640 medium was obtained from Corning, Inc. (Manassas, VA, USA).Enzyme-linked immunosorbent assay (ELISA) kits for assaying IL-6 were obtained from MyBioSource, Inc. (San Diego, CA, USA) and used per the manufacturer's instructions.Salmonella minnesota LPS was obtained from Sigma-Aldrich, Inc. (St Louis, MO, USA) and was used at a 2.5 µg/mL final concentration in PBS.Lymphocyte Separation Medium, density: 1.077 gms/mL (LSM TM , MP Biomedicals, Solon, OH, USA) was used for cell separation procedures (see below).
Cell separation procedures.Approximately 5 mL of heparinized chicken blood was layered onto an equal volume of LSM TM and was centrifuged at 400× g for 30 min.Four cell layers appeared post-centrifugation: a broad upper layer, a thin middle layer, a bottom broad layer, and a packed pellet of red cells at the bottom of the tube.Each of the top 3 layers were removed by pipetting, transferred to separates tubes, and resuspended in 10 mL of PBS.Cells were pelleted by centrifugation for 10 min at room temperature (400× g) and the pellets again resuspended in 10 mL of PBS.A second pelleting was followed by resuspension in 2 mL RPMI for subsequent testing.
Innate immunity whole blood assay procedure.Three to five mL of heparinized blood was collected from each bird, transferred into a 15 mL tube and kept at room temperature.The blood was immediately diluted 1:1 with RPMI 1640 medium (i.e., one part blood and one part medium) and inverted to thoroughly mix the blood with the medium.The sample was equally divided into two tubes.LPS (prepared in phosphate buffered saline (PBS)) was added to one of the tubes to achieve a final concentration of 2.5 µg/mL of LPS.An equivalent volume of PBS was added to the corresponding control tube.The tubes were then placed on a rotating mixer inside a 37 • C incubator.Prior to placing the tubes into the 37 • C incubator, a 0.5 mL sample was removed from each tube.Subsequent 0.5 mL samples were removed from each tube 2, 4, 24, and 48 h after placement into the 37 • C incubator.The 0.5 mL samples were delivered into 2 mL conical microcentrifuge tubes and centrifuged for 2 min at 5000× g.Following centrifugation, the supernatants were removed, and two individual IL-6 ELISAs were performed on each of the supernatants per the manufacturer's instructions.Briefly, 100 µL of sample was delivered into a well and incubated for 2 h at 37 • C. The liquid was removed and 100 µL of Detection Reagent A (MyBioSource, Inc.) was added and incubated at 37 • C for 1 h.The liquid was removed and washed three times with a wash buffer (MyBioSource, Inc.).Following washing, the residual wash buffer was removed by inverting and blotting the well onto a paper towel.One hundred microliters of Detection Reagent B (MyBioSource, Inc.) was added to each well and incubated for 1 h.The wells were again washed with wash buffer 5 times as above.Ninety microliters of Substrate Solution (MyBioSource, Inc.) was added and incubated for 10-20 min at 37 • C and protected from light.This was followed by adding 50 µL of Stop Solution (MyBioSource, Inc.) to the well and determining the optical density (OD) on a microplate reader set to 450 nm.IL-6 positive control samples, provided by the manufacturer, were also assayed to provide a standard curve.The control samples contained 0, 250, 500, and 1000 picograms (pgs) of IL-6 per mL.Estimates of IL-6 amounts Poultry 2024, 3 202 in the whole blood samples were extrapolated from the standard curve provided by the control sample OD readings.
Innate immunity assay with selected cell populations.Following the cell separation procedure described above, the resulting layers contained within the separation medium were harvested and washed with RPMI 1640, and direct smear slides were prepared and stained with Diff Quik stain (Fisher Scientific, Waltham, MA, USA) for light microscopy evaluation.The cells contained within the layers were evaluated for IL-6 production by ELISA in the same way as the whole blood assay described above using a 24-h activation time.
Experimental design for trial 1.Approximately 3 mL of blood was collected by the wing vein method from each of 5 female and 5 male SPF adult chickens (see above) into heparin-containing syringes.The blood samples were diluted 1:1 with RPMI 1640 medium and aliquoted into 2 separate tubes.To each tube, the diluted blood was activated with either LPS or with PBS (see above).At 0, 2-, 4-, 24-and 48-h post-activation, 500 µLs of blood was withdrawn from each sample at each time point.The blood was assayed for IL-6 by ELISA (see above) with each sample replicated once.The ODs of the IL-6 ELISA results were used to provide a more accurate assessment because IL-6 values are extrapolated from control standards and therefore are less precise.
Experimental design for trial 2. From one male and one female SPF bird, approximately 5 mL of blood was collected by the wing-vein method.White blood cells were separated by using LSM TM (see above).Cell counts were performed on the resulting cell layers and aliquoted into tubes at 10 5 cells/mL.The aliquots were activated with either PBS or LPS.At 24-h post-activation, 100 µL samples were removed and assayed for IL-6 using IL-6 ELISA kits (see above).Four replications per treatment were performed.
Experimental design for trial 3. Approximately 4 mL of blood was collected in heparin from one male bird and was diluted 1:1 with RPMI 1640 medium (as described above).The sample was aliquoted into three 2 mL tubes.To each tube, either PBS, LPS, or phytohemagglutinin A (PHA) was added.The PBS and LPS were added to the tubes as described above.One hundred microliters of PHA (Gibco, Inc., Grand Island, NY, USA) at a concentration of 100 µg/mL was added to the third sample.The samples were placed on a rotational mixer and incubated for 24 h (i.e., activation time).Following activation, the samples were processed for IL-6 using an ELISA kit as described above using four replicates per sample.
Statistical analysis.Descriptive analyses were carried out for the ODs of LPS activated cells, OD LPS, and the ODs obtained from the corresponding negative control PBS-only activated cells, OD PBS .Student's t-test was used to compare the within-group and betweengroup difference of group means of OD LPS and OD PBS at different time points.The data from all birds at each activation time interval were combined for statistical analysis.The group means of OD LPS and OD PBS, to determine if LPS activation was affected by sex factor, was also examined.All data analyses were implemented in Stata 16.1 (Stata Corp., College Station, TX, USA).
Activation index.An activation index was calculated from the difference in OD readings of IL-6 ELISA between the PBS-and LPS-group (or other compounds being evaluated) means divided by the PBS group mean as follows: [(Ave OD LPS − Ave OD PBS )/Ave OD PBS ] × 100%.

Results and Discussion
The results of trial 1 are displayed in Table S1 (see Supplementary Material) and Figure 1.IL-6 levels in all samples (i.e., PBS and LPS activated) diminished over time (see Figure 1).When compared to the PBS controls, IL-6 released by LPS activated cells were statistically significant (p < 0.05) at the 4-and 24-h post-activation intervals.The IL-6 levels present in the PBS groups are considered background levels of IL-6 and may have been caused by the stress of blood collection or other unknown causes.In studies involving rats which had been stimulated to produce IL-6, there were background IL-6 levels at time 0 prior to stimulation, and the half-life of IL-6 in rats was calculated to be 15.5 h [6].
The IL-6 half-life values for chickens were not found.However, as can be observed in Figure 1, the IL-6 level of the PBS control (background) at 24-h post-activation appears to be at a minimal level and was not significantly different than the IL-6 levels of the PBS controls at 48-h post-activation.The data suggest that those cells activated with LPS begin IL-6 production immediately; however, the largest difference between the PBS and LPS activated samples occurred at the 24-h post-activation sampling interval as indicated by the activation indices displayed in Figure 2. The 24-h post-activation time interval represents the period in which the IL-6 background level is at its minimum and the IL-6 production from LPS activated cells has peaked.The activation index was calculated by subtracting the LPS activation value from the PBS value (unactivated control) and then dividing the result by the PBS value and expressing the result as a percentage.The activation index provides a method for assessing the magnitude of the innate immune response and may prove useful when evaluating and comparing innate immune responses under different conditions.For example, when exploring and/or comparing substances and/or vaccine strategies that may enhance or suppress innate immunity (see Trial 3 below) or conditions (e.g., physiological stress caused by heat or dietary imbalances) that may affect the innate immune response.Based on these results, it was felt that an LPS activation time of 24 h was the most optimal time for the whole blood assay.
Poultry 2024, 3, FOR PEER REVIEW 4 which had been stimulated to produce IL-6, there were background IL-6 levels at time 0 prior to stimulation, and the half-life of IL-6 in rats was calculated to be 15.5 h [6].The IL-6 half-life values for chickens were not found.However, as can be observed in Figure 1, the IL-6 level of the PBS control (background) at 24-h post-activation appears to be at a minimal level and was not significantly different than the IL-6 levels of the PBS controls at 48-h post-activation.The data suggest that those cells activated with LPS begin IL-6 production immediately; however, the largest difference between the PBS and LPS activated samples occurred at the 24-h post-activation sampling interval as indicated by the activation indices displayed in Figure 2. The 24-h post-activation time interval represents the period in which the IL-6 background level is at its minimum and the IL-6 production from LPS activated cells has peaked.The activation index was calculated by subtracting the LPS activation value from the PBS value (unactivated control) and then dividing the result by the PBS value and expressing the result as a percentage.The activation index provides a method for assessing the magnitude of the innate immune response and may prove useful when evaluating and comparing innate immune responses under different conditions.For example, when exploring and/or comparing substances and/or vaccine strategies that may enhance or suppress innate immunity (see Trial 3 below) or conditions (e.g., physiological stress caused by heat or dietary imbalances) that may affect the innate immune response.Based on these results, it was felt that an LPS activation time of 24 h was the most optimal time for the whole blood assay.Statistical analysis of the data contained in Table S1 revealed that the within-group variation of ODs of PBS and LPS groups at the 0-, 2-, 4-, and 24-h time intervals was rather pronounced (Figure S1, see supplementary material).However, by the 24-h activation interval, the median values (as indicated by the lines contained within the bars in Figure S1) of the LPS OD groups had stabilized and were no longer decreasing.The variation of the PBS ODs achieved their minimal level by the 24-h activation interval.The 24-h activation indices of hens were compared to those of roosters and found not to be statistically significantly different (p > 0.18).In this study 10 birds (5 hens and 5 roosters) were used to obtain data.Although there was considerable variation between individual birds, statistical significance between the PBS and LPS IL-6 values was achieved at the 4-and 24-h activation time intervals, with the 24-h activation interval having the highest activation index.In view of these findings and analysis, it is suggested that to achieve the most appropriate interpretation of results, the whole blood innate immune assay should be conducted on a group-level of birds rather than on an individual bird-level.Statistical analysis of the data contained in Table S1 revealed that the within-group variation of ODs of PBS and LPS groups at the 0-, 2-, 4-, and 24-h time intervals was rather pronounced (Figure S1, see supplementary material).However, by the 24-h activation interval, the median values (as indicated by the lines contained within the bars in Figure S1) of the LPS OD groups had stabilized and were no longer decreasing.The variation of the PBS ODs achieved their minimal level by the 24-h activation interval.The 24-h activation indices of hens were compared to those of roosters and found not to be statistically significantly different (p > 0.18).In this study 10 birds (5 hens and 5 roosters) were used to obtain data.Although there was considerable variation between individual birds, statistical significance between the PBS and LPS IL-6 values was achieved at the 4-and 24-h activation time intervals, with the 24-h activation interval having the highest activation index.In view of these findings and analysis, it is suggested that to achieve the most appropriate interpretation of results, the whole blood innate immune assay should be conducted on a group-level of birds rather than on an individual bird-level.
Trial 2 was performed to identify which cell type may be responsible for IL-6 liberation.One rooster and one hen were selected to ensure there were no differences attributed to sex.The whole blood cell separation using the LSM TM medium resulted in erythrocytes being packed into a tight layer on the bottoms of the tubes with cell layers forming in the fluid above.Three layers were visualized and harvested: top, middle, and lower.The results from LPS activation of the cells from these layers with subsequent production of IL-6 are displayed in Table S2 (see supplementary material) and Figure 3.The 24-h activation indices for each of the cell layers are provided in Table S2 and Figure S2 (see supplementary material).As displayed in Figure 3, the highest IL-6 levels occurred in the top cell layer, with the lower layer also having significant IL-6 levels but lower than the top cell layer.This was observed with the separated cells from the hen and from the rooster.Figure 3 is a combination of the data from both birds.Images of cells from the three different cell layers are displayed in Figure 4.The three cell layers obtained with the LSM TM medium were not clearly distinct layers as one might observe when isolating viruses in an isopycnic gradient.Rather, the layers were hazy or cloudy in appearance, indicating that there were no single cells of one type (i.e., having the same buoyant density) contained in the layers.Cytological examination indicated the peripheral blood monocytes were in greatest numbers in the top layer and although present in the other layers, were far fewer in number.Additionally, it was noted that the peripheral blood monocytes contained within the top layer had formed aggregates (see Figure 4A), whereas peripheral blood Trial 2 was performed to identify which cell type may be responsible for IL-6 liberation.One rooster and one hen were selected to ensure there were no differences attributed to sex.The whole blood cell separation using the LSM TM medium resulted in erythrocytes being packed into a tight layer on the bottoms of the tubes with cell layers forming in the fluid above.Three layers were visualized and harvested: top, middle, and lower.The results from LPS activation of the cells from these layers with subsequent production of IL-6 are displayed in Table S2 (see supplementary material) and Figure 3.The 24-h activation indices for each of the cell layers are provided in Table S2 and Figure S2 (see supplementary material).As displayed in Figure 3, the highest IL-6 levels occurred in the top cell layer, with the lower layer also having significant IL-6 levels but lower than the top cell layer.This was observed with the separated cells from the hen and from the rooster.Figure 3 is a combination of the data from both birds.Images of cells from the three different cell layers are displayed in Figure 4.The three cell layers obtained with the LSM TM medium were not clearly distinct layers as one might observe when isolating viruses in an isopycnic gradient.Rather, the layers were hazy or cloudy in appearance, indicating that there were no single cells of one type (i.e., having the same buoyant density) contained in the layers.Cytological examination indicated the peripheral blood monocytes were in greatest numbers in the top layer and although present in the other layers, were far fewer in number.Additionally, it was noted that the peripheral blood monocytes contained within the top layer had formed aggregates (see Figure 4A), whereas peripheral blood monocytes observed in the other layers were far less numerous and typically occurred as individual cells.The IL-6 production following LPS activation in this report is attributed to the peripheral blood monocytes, which is in congruence with reports by others [2,7,8].The middle layer was composed predominantly of lymphocytes and thrombocytes (see Figure 4B).The lower layer contained large numbers of granulocytes along with monocytes (see Figure 4C).The IL-6 levels observed in the middle and bottom layers were presumed to be from monocytes that were observed in these layers.It has been reported that LPS-stimulated thrombocytes upregulate IL-6 genes that lead to the production of active IL-6 [9].It is recognized that different assays and approaches were used in this study; however, the data presented here do not support the conclusions of the aforementioned study, since the middle layer, which was predominantly composed of lymphocytes and thrombocytes, had significantly lower IL-6 levels compared to the top layer where high numbers of monocytes were present.
Poultry 2024, 3 205 sumed to be from monocytes that were observed in these layers.It has been reported that LPS-stimulated thrombocytes upregulate IL-6 genes that lead to the production of active IL-6 [9].It is recognized that different assays and approaches were used in this study; however, the data presented here do not support the conclusions of the aforementioned study, since the middle layer, which was predominantly composed of lymphocytes and thrombocytes, had significantly lower IL-6 levels compared to the top layer where high numbers of monocytes were present.The results of trial 3 are displayed in Figure S3 (see Supplementary Material).Trial 3 was a preliminary trial conducted to illustrate the applicability and usefulness of the whole blood innate immune assay using the activation index.In this preliminary trial, we used PHA as an innate immunity inducer and compared it with LPS.PHA was selected because of its reported immunostimulatory effects and its ability to potentially induce an acquired cell-mediated response and potentially an innate immune response [10,11].As can be observed in Figure S3, PHA had an activation index of 34%, indicating that an innate immune response did occur.However, the PHA response was much less robust than that achieved by LPS with an activation index of 90%.Again, this trial was included as an example of how the whole blood innate immune assay might have a practical application.However, if a more comprehensive and extensive evaluation of PHA as an innate immunity inducer were needed, then using a group level of birds (as described and recommended in trial 1 above) would be indicated and appropriate.Exploring and using compounds to induce the innate immune response may be important for vaccine strategies and generating trained innate immunity.
Figure 5 is a pictorial diagram summarizing the methodology used in the whole blood innate immunity assay.The procedure used whole blood collected in heparin.Other anticoagulants may be used, but heparin was readily available and worked well.The amount of whole blood collected in this study (i.e., trial 1 above) was 3-5 mL from adult chickens.However, the amount of blood collected can vary and may be determined by the size of the bird.For example, the authors have used as little as 0.3 mL (i.e., 300 µL) when collecting whole blood from day-old chicks.The whole blood was processed as soon as possible but was never kept at room temperature for more than 30 min before it was diluted 1:1 with RPMI 1640 medium.Following dilution with RPMI 1640 and mixing, the blood sample was aliquoted into two tubes.It was found that "snap cap" 2 mL microcentrifuge tubes worked well.These tubes are inexpensive and readily available; they were easily placed on a small (low-speed) rotary mixer and then placed into a 37 • C incubator for 24 h (i.e., activation interval).Following activation, the tubes were removed from the incubator, placed into a microcentrifuge and subjected to 5000× g for 2 min.The supernatants were removed and used in the IL-6 ELISA kits to determine IL-6 values.Note that the OD readings were used to determine statistical significance and the activation index.The LPS concentration used in these studies was 2.5 µg/mL final volume.This concentration was determined by previous reports in the literature.Rattanasrisomporn et al. [8] demonstrated highest IL-6 liberation from peripheral blood monocytes (PBMCs) using 1 µg/mL of LPS.Verwoolde et.al. [2,12] stimulated PBMCs with 10 µg/mL of LPS.This study used 2.5 µg/mL of LPS with whole blood and found adequate stimulation without signs of cell toxicity.

Conclusions
A whole blood assay to determine the innate immune response using LPS with a 24h activation time interval produced statistically significant levels of IL-6 when compared to PBS controls.An activation index was calculated to demonstrate the maximum effects of LPS activation.There was pronounced variation between individual birds.Variation between birds was least at the 24-h post-activation interval with no differences between the activation indices of hens and roosters.Given the variation observed between birds in this study, it is recommended to use groups of birds and interpret the results by combining the data to achieve optimum results.The primary cell type for IL-6 production was identified to be the peripheral blood monocyte.The whole blood method proved to be accurate, minimally invasive, easy to perform, used conventional laboratory equipment, and induced minimal stress on the bird.

Poultry 2024, 3 204 5 Figure 2 .
Figure 2. Graphical summation of data from Trial 1/Table S1 displaying activation indices from all birds at activation time intervals.

Poultry 2024, 3 , 7 Figure 4 . 9 Figure 5 .
Figure 4. (A).Monocytes are the predominant cell type within the top layer, characterized by oval-to reniform-shaped eccentric nuclei and abundant foamy cytoplasm (arrows).Degenerating monocytes Graphical summation of data from Trial 1/TableS1displaying activation indices from all birds at activation time intervals.