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. 2024 Jan 22:14:1319947.
doi: 10.3389/fimmu.2023.1319947. eCollection 2023.

Canine diabetes mellitus demonstrates multiple markers of chronic inflammation including Th40 cell increases and elevated systemic-immune inflammation index, consistent with autoimmune dysregulation

Gisela Vaitaitis  1 , Tracy Webb  2 , Craig Webb  2 , Christina Sharkey  3 , Steve Sharkey  3 , Dan Waid  4 , David H Wagner  1   4
Affiliations

Canine diabetes mellitus demonstrates multiple markers of chronic inflammation including Th40 cell increases and elevated systemic-immune inflammation index, consistent with autoimmune dysregulation

Gisela Vaitaitis et al. Front Immunol. .

Abstract

Introduction: Canine diabetes mellitus (CDM) is a relatively common endocrine disease in dogs. Many CDM clinical features resemble human type 1 diabetes mellitus (T1DM), but lack of autoimmune biomarkers makes calling the disease autoimmune controversial. Autoimmune biomarkers linking CDM and T1DM would create an alternative model for drug development impacting both human and canine disease.

Methods: We examined peripheral blood of diagnosed CDM dog patients comparing it to healthy control (HC) dogs. Dogs were recruited to a study at the Colorado State University Veterinary Teaching Hospital and blood samples collected for blood chemistry panels, complete blood counts (CBC), and immunologic analysis. Markers of disease progression such as glycated albumin (fructosamine, the canine equivalent of human HbA1c) and c-peptide were addressed.

Results: Significant differences in adaptive immune lymphocytes, innate immune macrophages/monocytes and neutrophils and differences in platelets were detected between CDM and HC based on CBC. Significant differences in serum glucose, cholesterol and the liver function enzyme alkaline phosphatase were also detected. A systemic immune inflammation index (SII) and chronic inflammation index (CII) as measures of dynamic changes in adaptive and innate cells between inflammatory and non-inflammatory conditions were created with highly significant differences between CDM and HC. Th40 cells (CD4+CD40+ T cells) that are demonstrably pathogenic in mouse T1DM and able to differentiate diabetic from non-diabetic subjects in human T1DM were significantly expanded in peripheral blood mononuclear cells.

Conclusions: Based on each clinical finding, CDM can be categorized as an autoimmune condition. The association of significantly elevated Th40 cells in CDM when compared to HC or to osteoarthritis, a chronic but non-autoimmune disease, suggests peripheral blood Th40 cell numbers as a biomarker that reflects CDM chronic inflammation. The differences in SII and CII further underscore those findings.

Keywords: Th40 cells; autoimmune; canine diabetes mellitus; chronic inflammation; inflammatory index.

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Conflict of interest statement

DHW is co-founder of OP-T, LLC a biotech company. DW is employed by Op-T, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Blood chemistry panels were collected on CDM and HC dogs. Blood was sent to Antech Diagnostic Laboratory for analysis. (A) Serum glucose is a standard observation, (B) Serum cholesterol is not obtained on most dogs but is included in diabetic dogs due to risk of cardiovascular disease. (C) Alkaline Phosphatase, (D) Fructosamine, and (E) C-Peptide The green lines indicate established normal range. Statistics were done using GraphPad Prism using a two-tailed, unpaired t-test analysis.
Figure 2
Figure 2
Data are taken from complete blood counts done on each dog; the absolute number per microliter is reported. (A) Lymphocytes, (B) Monocytes, (C) Neutrophils, (D) Platelets and (E) mean platelet volume (MPV) were done. MPVs were done only on the CDM dogs. Like serum cholesterol, MPV is a special request in the CBC. Statistics were done using GraphPad Prism using a two-tailed, unpaired t-test analysis.
Figure 3
Figure 3
Indices of Chronic Inflammation: (A) Neutrophil to lymphocyte ratio (NLR) is calculated by dividing the absolute number of neutrophils per microliter by the absolute number of lymphocytes per microliter of blood. (B) Monocyte to lymphocyte ratio (MLR) is the absolute number of monocytes divided by the absolute number of lymphocytes. (C) Platelet to lymphocyte (PLR) is the total number of platelets per microliter divided by 103 times absolute number of lymphocytes. (Platelets are reported as whole number x 10^3 per microliter). (D) Systemic Immune Inflammation Index (SII) is calculated by (total number of neutrophils) x (whole number of platelets/10^3)/whole number of lymphocytes. (E) Chronic Immune Inflammation Index (CII) is calculated by: (whole number of monocytes) x (whole number of platelets/10^3)/whole number of lymphocytes. Statistics were done from GraphPad Prism using a two-tailed, unpaired t-test analysis.
Figure 4
Figure 4
Th40 cell comparison in CD and HC. PBMC analyzed for CD3, CD4 and CD40 (Th40 cells). (A) Healthy controls and (B) Canine Diabetes were analyzed by dot plots to establish forward versus side scatter. Within the FSC x SSC population CD3+ cells are determined above background controls (grey solid histogram). Within the gated CD3+ population, contour plots of CD4 versus CD40 were determined. Th40 cells are defined as CD3+ then CD4+CD40+/total CD4+ cells (Q2/Q2 + Q3). (C) Th40 percentage in CD dogs compared to HC dogs. Statistics were done using Graph Pad Prism using a two-tailed, unpaired t-test analysis.
Figure 5
Figure 5
Inflammatory cytokine levels in CDM Th40 cells. PBMC from nine CDM and five healthy controls were analyzed. (A) Cells were stained for CD3 then gated against CD4 and CD40; dot plot is representative of CDM. Within the Th40 (CD4+CD40+) and Conventional T cell (CD4+CD40-) populations, intracellular levels of (B) IL-6 and (C) IFNγ were determined. Statistics were done from GraphPad Prism using a two-tailed, unpaired t-test analysis.
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