Coccidioidomycosis is an endemic disease in the southwestern US caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii.1,2 Infection is usually initiated through the inhalation of arthrospores present in the environment. After arthrospores transform into the spherule phase, a spectrum of disease, from subclinical infection to severe respiratory tract infection and/or disseminated disease, can occur.1,2 Approximately 60% of Coccidioides spp infections are subclinical in humans, and approximately 70% are suspected to be subclinical in dogs.3–5
Information regarding coccidioidomycosis in cats is sparse compared to that for dogs; however, cats have an increased frequency of disseminated disease (50% to 60%) compared to dogs (20% to 42%).6–10 Additionally, dermatologic lesions are a common manifestation of disseminated coccidioidomycosis in cats (43% to 56%), whereas the most common sites of dissemination in dogs include bones, joints, and lymph nodes.1,6–9 It is unknown whether the increased frequency of disseminated disease is associated with cats being diagnosed later in the stage of disease or whether there are differences in the host immune response to infection compared to dogs.6 Like humans and dogs, serologic tests to detect anti–Coccidioides spp immunoglobulin (Ig)-M and IgG antibodies remain a focal point in establishing a clinical diagnosis of coccidioidomycosis in cats. Regardless of species, coccidioidal antigen chitinase-1 (CTS1), also referred to as complement fixation antigens, is used to detect IgG reactivity.11
Stimulation of early immune response that manifests following exposure to Coccidioides spp has shown to be predictive of disease progression in humans.12–14 A cell-mediated response with a release of T-helper (Th)-1 cytokines, including interferon-γ (IFN-γ) and IL-2, has been associated with disease resolution and a good outcome.15,16 Neutralization of Coccidioides spp endospores relies on the release of the proinflammatory cytokines IFN-γ and tumor necrosis factor-α (TNF-α), and secretion of these cytokines by peripheral blood mononuclear cells has been shown to occur following in vitro whole-blood coccidioidal antigen stimulation.17–20 More recent studies14,21–23 in mice substantiate a role of Th17 cells and the release of IL-17 in long-term immunity. Conversely, a Th2 response has been associated with chronic disease.12,24 While the evaluation of immune response to Coccidioides spp has been explored in humans and, to a much lesser extent, in dogs, it has, to the authors’ knowledge, not been evaluated in cats. Understanding the early immune response to Coccidioides spp in cats may provide insight into the various clinical manifestations and progression of disease in this species.
The focus of the present study was to evaluate the pattern of cytokine expression in whole blood from healthy cats incubated with recombinant CTS1 (rCTS1105–310). Coccidioidal antigen chitinase-1 is a chitinase involved in spherule growth and endospore release, is immunogenic, and is used in serodiagnostic testing.25,26 The objectives of this study were therefore to (1) describe the ex vivo rCTS1105–310-stimulated cytokine profile in healthy cats and (2) determine the rCTS1105–310 concentration and incubation time that optimized cytokine concentrations. We hypothesized that rCTS1105–310 would stimulate Th1-related and proinflammatory cytokine production in the whole blood of healthy cats.
Methods
Animals
Six healthy, adult cats were enrolled in this study. All participants were privately owned cats of employees or students at Midwestern University. Cats lived entirely inside the owners’ residence. Inclusion criteria included healthy status based on unremarkable physical examination and review of a CBC, serum biochemistry profile, and, when possible, a urinalysis by a board-certified small-animal internist (JAJ; Antech Diagnostics). Included cats were also required to have negative anti–Coccidioides spp antibody (IgM and IgG) titer results measured with agar gel immunodiffusion at the time of enrollment (Antech Diagnostics). Exclusion criteria included prior medical illnesses or current administration of medications outside of standard monthly parasite preventatives within 60 days of enrollment. This study was approved by the IACUC at Midwestern University (IACUC #AZ-4009), and informed consent was obtained from all owners.
Sample collection and processing
Medical records were reviewed for each cat enrolled. The age, sex, weight, and breed were recorded. Blood samples were collected into blood tubes with lithium heparin as an anticoagulant, and study experiments were started within 1 hour of sample collection.
Coccidioidal antigen chitinase-1 preparation
Recombinant CTS1 is a truncated polypeptide encompassing the protein segment that is immunodominant in human serum. Recombinant CTS1 was expressed and purified as previously described.27 Briefly, the aa105–310 truncation of rCTS1 was cloned into a ligation-independent cloning vector pMCSG7 that contained a T7 promoter and N-terminal 6X His-tag.28 The plasmid was grown in BL21 Gold (Agilent) cells in Terrific Broth (Millipore) to optical density (OD)600 0.6 and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Thermo Fisher). Cells were collected and resuspended in 20 mM Tris (pH 8), 200 mM NaCl, 25 mM imidazole, and 5% glycerol with protease inhibitors and deoxyribonuclease. Cells were sonicated, and the soluble fraction was run on an immobilized nickel-affinity chromatography (HisTrap FF; GE Healthcare). Proteins were further purified by size-exclusion chromatography (Superdex 200; GE HealthCare) using a buffer of 20 mM Tris (pH 8), 200 mM NaCl, and 5% glycerol. Protein was concentrated if necessary, and concentration ion was determined by absorbance at 280 nm using molecular weight and the extinction coefficient. Samples at > 10 μM were aliquoted into thin-walled PCR tubes and flash frozen using liquid nitrogen.
Whole-blood cytokine stimulation and measurement
Heparinized whole blood was diluted 1:2 with RPMI culture medium (Thermo Fisher Scientific) containing 200 U of penicillin/mL and 200 mg of streptomycin/mL. The blood-RPMI mixture from each cat was then transferred to 96-well plates and incubated with rCTS1105–310 at 5 final well concentrations (1 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, and 75 µg/mL) or PBS as a vehicle control. Twelve wells were used per cat to reflect the 6 exposure conditions (ie, rCTS1105–310 or PBS) and 2 incubation times. Plates were incubated for 12 hours and 24 hours at 37 °C in 5% CO2 in the dark. Following incubation at both time points, plates were centrifuged (400 X g for 7 minutes) at 21 °C. The supernatant was collected and stored at −80 °C for batch analysis. For analysis, samples were thawed, and then TNF-α, IL-6, IL-12, IL-2, IL-4, IL-1β, IL-8, granulocyte macrophage CSF (GM-CSF), monocyte chemoattractant protein-1, and IFN-γ were measured with a feline cytokine–specific, multiplex bead–based assay (Milliplex MAP; EMD Millipore Corp) as described elsewhere.29 The median fluorescence intensity and cytokine concentration in each sample were measured in duplicate with appropriate manufacturer controls and associated data analysis software (Milliplex Analyst, version 5.1; EMD Millipore Corp). The lower limit of detection (LLD) for TNF-α, IL-2, IL-8, IL-12, and GM-CSF was 41.15 pg/mL. The LLD for IFN-γ, IL-4, and IL-6 was 205.76 pg/mL. The LLD for IL-1β and monocyte chemoattractant protein-1 was 102.88 l pg/mL and 1,235 pg/mL, respectively.
Statistical analysis
Statistical analyses were performed using statistical software (R, version 4.1; R Foundation for Statistical Computing). Descriptive continuous data, including age and weight, were normally distributed as determined by Shapiro-Wilk tests and presented as mean and SD. Descriptive data from the CBC were non-normally distributed and presented as median and IQR. When the measured cytokine concentration fell below the LLD, data were recorded at the LLD for statistical purposes. The statistical analysis used to investigate the effect of rCTS1105–310 concentration on cytokine by time (an analysis for 12 hours and 24 hours) was the Kruskal-Wallis test. For any statistically significant results between groups overall, the Dunn test for multiple comparisons with a P value adjustment using the Bonferroni method was conducted. The analysis for the effect of time on cytokine concentration by rCTS1105–310 concentration (an analysis for each rCTS1105–310 concentration of 1, 10, 25, 50, and 75 µg/mL + control) was the Wilcoxon signed-rank test with Bonferroni correction. The significance level was set at α < 0.05.
Results
Animals
Six healthy, adult domestic long- or shorthair cats (4 neutered males, 2 spayed females) ranging from 1 to 8 years of age (mean ± SD age, 3.3 ± 2.4 years) and weighing 4.4 to 8.8 kg (mean ± SD weight, 5.8 ± 1.8 kg) were enrolled in this study. Descriptive data from the CBC were WBCs (median, 8.8 X 103/µL; IQR, 7.0 to 12.7), neutrophils (median, 4,588/µL; IQR, 3,286.3 to 5,558.0), monocytes (median, 193.5/µL; IQR, 138.0 to 335.0), lymphocytes (median, 3,624.0/µL; IQR, 3,021.0 to 6,284.0), and eosinophils (median, 390/µL; IQR, 116.3 to 678.8).
Whole-blood cytokine stimulation and measurement
Concentrations of GM-CSF and IFN-γ were below the LLD in all samples at all timepoints, including the unstimulated controls and all concentrations of rCTS1105–310 at both the 12-hour and 24-hour incubation periods. Therefore, these 2 analytes were excluded from subsequent analyses.
For the analysis of rCTS1105–310 concentration by time on cytokine concentration, Kruskal-Wallis tests revealed a significant difference for IL-1β at both the 12-hour (P = .003) and 24-hour (P = .02) incubation times compared with control. Post hoc comparisons using the Dunn test with Bonferroni adjustment showed that the incubation of whole blood with rCTS1105–310 for 12 hours for IL-1β at 50 µg/mL (mean, 274.4 pg/mL; SD, 110.2; P = .02) and 75 µg/mL (mean, 298.4 pg/mL; SD, 110.7; P = .003) resulted in increased supernatant concentrations of IL-1β compared to control (mean, 102.9 pg/mL; SD, 0.0; Table 1). Similarly, results from the Dunn tests revealed that exposure with rCTS1105–310 for 24 hours for IL-1β at 50 µg/mL (mean, 340.3 pg/mL; SD, 129.7; P = .02) and 75 µg/mL (mean, 364.8 pg/mL; SD, 151.9; P = .01) increased IL-1β concentrations compared with control (mean, 110.5 pg/mL; SD, 18.7; Table 2). There were no other significant differences in cytokine concentrations for any of the other rCTS1105–310 concentrations at either the 12-hour or 24-hour incubation times (all P > .05).
Supernatant cytokine concentrations (picograms per milliliter) in 6 healthy cats following whole-blood incubation for 12 hours with various concentrations of recombinant coccidioidal antigen chitinase-1 (rCTS1)105–310 antigen or PBS control.
| 1 μg/mL (n = 6) | 10 μg/mL (n = 6) | 25 μg/mL (n = 6) | 50 μg/mL (n = 6) | 75 μg/mL (n = 6) | PBS control (n = 6) | P value | |
|---|---|---|---|---|---|---|---|
| IL-1β | .003 | ||||||
| Mean (SD) | 116.847 (14.770) | 213.250 (93.156) | 252.915 (114.996) | 274.352 (110.167) | 298.442 (110.706) | 102.880 (0.000) | |
| Range | 102.880–137.080 | 102.880–325.550 | 102.880–422.970 | 102.880–395.220 | 121.010–441.020 | 102.880 – 102.880 | |
| IL-2 | 1.00 | ||||||
| Mean (SD) | 49.492 (20.433) | 45.428 (10.480) | 47.822 (16.342) | 46.958 (14.227) | 44.525 (8.267) | 47.107 (14.591) | |
| Range | 41.150–91.200 | 41.150–66.820 | 41.150–81.180 | 41.150–76.000 | 41.150–61.400 | 41.150–76.890 | |
| IL-12 | .83 | ||||||
| Mean (SD) | 132.490 (59.299) | 129.395 (55.122) | 126.250 (50.343) | 138.413 (58.382) | 133.603 (59.718) | 118.260 (57.096) | |
| Range | 41.150–226.780 | 44.340–216.380 | 41.630–196.080 | 41.150–221.410 | 44.170–230.430 | 41.150–216.540 | |
| IL-4 | 1.00 | ||||||
| Mean (SD) | 379.423 (378.187) | 289.358 (180.307) | 322.325 (249.463) | 349.815 (300.416) | 311.955 (204.564) | 296.843 (213.658) | |
| Range | 205.760–1,147.000 | 205.760–655.810 | 205.760–829.200 | 205.760–959.540 | 205.760–722.870 | 205.760–732.860 | |
| IL-6 | .99 | ||||||
| Mean (SD) | 268.568 (153.648) | 244.838 (68.586) | 251.913 (102.294) | 257.193 (101.986) | 238.866 (67.295) | 239.005 (81.433) | |
| Range | 205.760–582.200 | 205.760–373.960 | 205.760–459.890 | 205.760–460.710 | 205.760–373.966 | 205.760–405.230 | |
| IL-8 | .40 | ||||||
| Mean (SD) | 923.180 (544.502) | 1,086.988 (785.533) | 996.940 (641.445) | 1,005.037 (641.713) | 1,084.312 (710.446) | 442.323 (637.907) | |
| Range | 408.270–1,734.000 | 467.690–2,449.000 | 434.030–2,095.000 | 418.680–2,043.000 | 498.790–2,169.000 | 41.150–1,647.000 | |
| MCP-1 | .97 | ||||||
| Mean (SD) | 2,531.167 (1,301.730) | 2,324.500 (899.517) | 2,429.667 (1,096.709) | 2,577.833 (1,138.479) | 2,332.167 (731.251) | 2,277.333 (1,053.958) | |
| Range | 1,235.000–4,969.000 | 1,235.000–3,906.000 | 1,235.000–4,443.000 | 1,235.000–4,672.000 | 1,235.000–3,402.000 | 1,235.000–4,237.000 | |
| TNF-α | .35 | ||||||
| Mean (SD) | 186.772 (150.228) | 201.650 (178.887) | 198.215 (133.989) | 244.010 (173.882) | 234.782 (155.734) | 107.218 (76.532) | |
| Range | 43.940–433.120 | 48.140–538.450 | 49.400–409.810 | 63.200–516.950 | 65.700–518.610 | 41.150–246.690 |
Data were analyzed using Kruskal-Wallis tests.
MCP = Monocyte chemoattractant protein. TNF = Tumor necrosis factor.
Supernatant cytokine concentrations (picograms per milliliter) in 6 healthy cats following whole-blood incubation for 24 hours with various concentrations of rCTS1105–310 antigen or PBS control.
| 1 μg/mL (n = 6) | 10 μg/mL (n = 6) | 25 μg/mL (n = 6) | 50 μg/mL (n = 6) | 75 μg/mL (n = 6) | PBS control (n = 6) | P value | |
|---|---|---|---|---|---|---|---|
| IL-1β | .02 | ||||||
| Mean (SD) | 237.035 (117.427) | 332.020 (177.406) | 319.135 (152.939) | 340.307 (129.719) | 364.805 (151.901) | 110.500 (18.665) | |
| Range | 102.880–435.370 | 102.880–538.420 | 102.880–507.020 | 132.740–466.590 | 173.250–585.350 | 102.880–148.600 | |
| IL-2 | 1.00 | ||||||
| Mean (SD) | 47.775 (16.228) | 49.145 (19.584) | 49.030 (19.302) | 47.107 (14.591) | 44.550 (8.328) | 51.313 (24.895) | |
| Range | 41.150–80.900 | 41.150–89.120 | 41.150–88.430 | 41.150–76.890 | 41.150–61.550 | 41.150–102.130 | |
| IL-12 | .93 | ||||||
| Mean (SD) | 139.510 (60.774) | 149.613 (67.362) | 138.307 (63.016) | 141.717 (55.966) | 140.813 (55.945) | 119.398 (44.697) | |
| Range | 42.700–233.000 | 41.150–242.300 | 41.150–239.640 | 48.590–222.400 | 53.070–223.400 | 41.150–165.810 | |
| IL-4 | 1.00 | ||||||
| Mean (SD) | 326.487 (234.693) | 373.575 (322.174) | 361.263 (303.530) | 328.442 (231.857) | 298.950 (168.694) | 352.292 (299.073) | |
| Range | 205.760–800.280 | 205.760–1,024.000 | 205.760–974.920 | 205.760–795.070 | 205.760–635.680 | 205.760–959.420 | |
| IL-6 | .36 | ||||||
| Mean (SD) | 315.530 (96.031) | 322.163 (127.382) | 277.592 (122.626) | 258.953 (92.035) | 237.458 (52.479) | 269.592 (135.541) | |
| Range | 205.760–454.190 | 205.760–545.810 | 205.760–515.480 | 205.760–432.930 | 205.760–330.120 | 205.760–543.820 | |
| IL-8 | .69 | ||||||
| Mean (SD) | 1,606.440 (1,068.187) | 1,554.570 (1,223.085) | 1,704.215 (1,286.326) | 1,681.827 (1,188.246) | 1,661.225 (1,126.172) | 913.337 (1,027.100) | |
| Range | 539.430–3,233.000 | 487.850–3,725.000 | 488.650–3,338.000 | 628.040–3,453.000 | 617.020–3,488.000 | 90.600–2,773.000 | |
| MCP-1 | .98 | ||||||
| Mean (SD) | 2,402.500 (1,056.953) | 2,519.333 (909.334) | 2,484.000 (862.447) | 2,546.500 (1,034.421) | 2,446.167 (852.354) | 2,438.833 (1,237.086) | |
| Range | 1,235.000–4,309.000 | 1,235.000–4,002.000 | 1,235.000–3,886.000 | 1,235.000–4,384.000 | 1,235.000–3,866.000 | 1,235.000–4,760.000 | |
| TNF-α | .90 | ||||||
| Mean (SD) | 140.848 (84.060) | 170.932 (139.397) | 157.148 (112.497) | 153.375 (105.793) | 144.680 (94.065) | 112.988 (106.986) | |
| Range | 43.940–261.370 | 52.330–379.780 | 53.170–307.640 | 59.030–303.300 | 61.540–303.950 | 41.150–322.920 |
Data were analyzed using Kruskal-Wallis tests.
Wilcoxon signed-rank tests were performed to investigate the effect of time on cytokine concentration by rCTS1105–310 concentration and revealed that for the 1-µg/mL rCTS1105–310 concentration, IL-1β concentrations were greater after incubation for 24 hours (mean, 237.0 pg/mL; SD, 117.4) than 12 hours (mean, 116.8 pg/mL; SD, 14.8; P = .04; Table 3). There were no other significant differences in cytokine concentrations for any other rCTS1105–310 concentration between 12 hours and 24 hours (all P > .05; Supplementary Tables S1–S5). Regardless of incubation time, the exposure of cells to PBS resulted in IL-1β concentrations below the LLD in most cats. Only 1 cat had measurable IL-1β (148.6 pg/mL) when cells were incubated with PBS.
Supernatant cytokine concentrations (picograms per milliliter) in 6 healthy cats following whole-blood incubation of rCTS1105–310 antigen at 1 µg/mL for 12 hours versus 24 hours.
| 12 hours (n = 6) | 24 hours (n = 6) | P value | |
|---|---|---|---|
| IL-1β | |||
| Mean (SD) | 116.847 (14.770) | 237.035 (117.427) | .04 |
| Range | 102.880–137.080 | 102.880–435.370 | |
| IL-2 | |||
| Mean (SD) | 49.492 (20.433) | 47.775 (16.228) | 1.00 |
| Range | 41.150–91.200 | 41.150–80.900 | |
| IL-12 | |||
| Mean (SD) | 132.490 (59.299) | 139.510 (60.774) | .48 |
| Range | 41.150–226.780 | 42.700–233.000 | |
| IL-4 | |||
| Mean (SD) | 379.423 (378.187) | 326.487 (234.693) | .93 |
| Range | 205.760–1,147.000 | 205.760–800.280 | |
| IL-6 | |||
| Mean (SD) | 268.568 (153.648) | 315.530 (96.031) | .16 |
| Range | 205.760–582.200 | 205.760–454.190 | |
| IL-8 | |||
| Mean (SD) | 923.180 (544.502) | 1,606.440 (1,068.187) | .24 |
| Range | 408.270–1,734.000 | 539.430–3,233.000 | |
| MCP-1 | |||
| Mean (SD) | 2,531.167 (1,301.730) | 2,402.500 (1,056.953) | 1.00 |
| Range | 1,235.000–4,969.000 | 1,235.000–4,309.000 | |
| TNF-α | |||
| Mean (SD) | 186.772 (150.228) | 140.848 (84.060) | .52 |
| Range | 43.940–433.120 | 43.940–261.370 |
Data were analyzed using Wilcoxon signed rank tests.
Discussion
Whole-blood stimulation with immunogenic antigens is a straightforward and rapid method to evaluate the response to various stimuli, which can aid in characterizing the associated innate and adaptive immune responses.30–34 Stimulation with pathogen-associated molecular patterns and subsequent evaluation of cytokines and other inflammatory markers has been performed previously in cats to characterize the cytokine signature.29,30 These studies have identified cytokine signatures in response to whole-blood stimulation with bacterial pathogen-associated molecular patterns, including lipopolysaccharide, lipoteichoic acid, and peptidoglycan; however, to the authors’ knowledge, this represents the first report of whole-blood stimulation with a single recombinant coccidioidal antigen in cats.29,30
Incubation of whole blood with various concentrations of rCTS1105–310 for 12 and 24 hours was performed to determine the minimum concentration and incubation time to stimulate cytokine production. A significant difference in cytokine stimulation between the 12-hour and 24-hour incubation periods was not observed, except for IL-1β, where a significantly increased concentration was noted following the 24-hour incubation only when stimulated with rCTS1105–310 at a concentration of 1 µg/mL. These findings suggest that while a 12-hour incubation period may be sufficient, 24 hours may be ideal. The concentration of rCTS1105–310 used for antigenic stimulation was only significant for IL-1β production. Specifically, rCTS1105–310 concentrations of 50 µg/mL and 75 µg/mL for 12 hours increased IL-1β production compared to control. Similarly, an rCTS1105–310 concentration of 75 µg/mL for 24 hours also increased supernatant concentrations of IL-1β. Taken together, our results suggest that the exposure of cells to rCTS1105–310 at 75 µg/mL for 24 hours is likely optimal for the production of IL-1β in cats. The small sample population of this study was an important limitation that may have impeded the identification of true significant differences (a type 2 error). As with most exploratory studies, obtaining estimates of SD to plan future studies in this area of research was still valuable despite very likely being underpowered.
Changes in ex vivo coccidioidal antigen–stimulated cytokine concentrations in human patients and dogs with and without coccidioidomycosis are variable; however, an increase in TNF-α, GM-CSF, and IFN-γ seem to be consistently increased in both humans and dogs. In a study conducted by Ampel et al,20 whole-blood stimulation with the coccidioidal antigen T27K was performed on blood samples from humans with a recent diagnosis of coccidioidomycosis and 2 control groups of patients without a clinical diagnosis of coccidioidomycosis, including an immune and nonimmune control group. Increased concentrations of GM-CSF, IL-1β, IL-2, IFN-γ, IL-13, and TNF-α were observed following whole-blood stimulation in patients recently diagnosed with pulmonary coccidioidomycosis and immune control subjects when compared to healthy nonimmune subjects.20 The nonimmune human subjects failed to exhibit a significant increase in proinflammatory cytokine concentrations. A recent study conducted by Jaffey et al34 in dogs evaluated ex vivo changes in cytokine concentrations following whole-blood stimulation with rCTS1105–310 in cases newly diagnosed with coccidioidomycosis and healthy controls. Increased supernatant concentrations of TNF-α, IL-6, GM-CSF, IL-8, and IL-10 were noted in control dogs and those with coccidioidomycosis.
Our study had several limitations that must be considered. As mentioned earlier, the small sample population with experiments under various different conditions led to limited observations and likely being underpowered to detect differences in all scenarios. Data obtained from this study will be instrumental in sample size calculation and study design in future studies investigating rCTS1105–310 as a coccidioidal antigen in cats. There remains a void in definitively differentiating between immune and nonimmune in client-owned domestic pets (ie, cats and dogs), which was a limitation of our study and the recently published Jaffey et al34 study that focused on dogs. Control pets in both studies were healthy and seronegative for Coccidioides spp antibodies, but this does not exclude the possibility of a previous subclinical infection. Utilization of healthy cats from a nonendemic area or specific pathogen-free cats would provide more clarity on whether immune status for Coccidioides spp affects ex vivo cytokine production following antigen stimulation.
The inclusion of only healthy cats was purposefully strict in this exploratory study as to avoid the inclusion of confounding variables that may have affected cytokine production, such as comorbid disorders or medication administration. Whole-blood cultures, rather than isolated immune cell types, were used to investigate cytokine production because this microenvironment allowed for cellular reactions to occur in a more physiologic milieu, possibly improving the clinical relevance of these ex vivo results. Many of the cytokines evaluated in our study were below the LLD regardless of whether samples were stimulated or not. This may be a true representation of cytokine production in cats with rCTS1105–310 stimulation or a potential limitation of the feline-specific, magnetic bead–based multiplex immunoassay.29 Most cytokines were below the LLD in a similarly designed ex vivo study in healthy cats using other antigenic stimulants (ie, lipopolysaccharide, lipoteichoic acid, peptidoglycan) and the same multiplex immunoassay.29 Future studies that focus on evaluating the cytokine immune profile in coccidioidal antigen–stimulated immune cells from cats may consider using PCR to detect cytokine mRNA transcripts or flow cytometry for the identification of intracellular cytokine concentration. Recombinant CTS1105–310 is a truncated polypeptide similar in structure to chitinase-1, a protein utilized by Coccidioides spp organisms to weaken the spherule cell wall before endospore release. Coccidioidal antigen chitinase-1 antigen has long been integral in the serodiagnostics of coccidioidomycosis in various species; however, the magnitude of its immunogenicity and specific signaling pathways remain unknown. Therefore, it is unclear whether the cytokine signature identified in this study was the result of a yet-to-be-determined signaling pathway or nonspecific antigenic stimulation. Additional research is needed to further classify this response. Lastly, the antigen used for stimulation experiments was a single recombinant polypeptide with no carbohydrate (sugar) moieties. Cytokine results may have been more similar to mouse and human patient studies if a more complex antigen were used to stimulate the cells from cats. Undoubtedly, additional work is needed to optimize this assay in domestic pets, including the antigens used, incubation time, and cytokines measured. The overarching goal with data from these future studies will be to better understand the immune response to Coccidioides spp in cats that may fill gaps in our comprehension of circumstances that surround infection susceptibility, severity, and progression to disseminated disease.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This study was partially funded by National Institute of General Medical Sciences grant No. 2R35128653.
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