Osteomyelitis is associated with increased anti-inflammatory response and immune exhaustion

Abstract

Introduction: Osteomyelitis (OMS) is a bone infection causing bone pain and severe complications. A balanced immune response is critical to eradicate infection without harming the host, yet pathogens manipulate immunity to establish a chronic infection. Understanding OMS-driven inflammation is essential for disease management, but comprehensive data on immune profiles and immune cell activation during OMS are lacking.

Methods: Using high-dimensional flow cytometry, we investigated the detailed innate and adaptive systemic immune cell populations in OMS and age- and sex-matched controls.

Results: Our study revealed that OMS is associated with increased levels of immune regulatory cells, namely T regulatory cells, B regulatory cells, and T follicular regulatory cells. In addition, the expression of immune activation markers HLA-DR and CD86 was decreased in OMS, while the expression of immune exhaustion markers TIM-3, PD-1, PD-L1, and VISTA was increased. Members of the T follicular helper (Tfh) cell family as well as classical and typical memory B cells were significantly increased in OMS individuals. We also found a strong correlation between memory B cells and Tfh cells.

Discussion: We conclude that OMS skews the host immune system towards the immunomodulatory arm and that the Tfh memory B cell axis is evident in OMS. Therefore, immune-directed therapies may be a promising alternative for eradication and recurrence of infection in OMS, particularly in individuals and areas where antibiotic resistance is a major concern.

Keywords: T follicular helper cells; immune exhaustion; infection; inflammation; musculoskeletal immunology; osteomyelitis.

Figure 1

Elevated Treg frequency in OMS subjects. (A) T cell frequencies (CD3⁺, CD4⁺, CD8⁺, and Tregs). Frequencies of (B) naïve, (C) effector memory, (D) central memory, (E) TEMRA, (F) activated, (G) Tscm, (H) senescence, and (I) PD1⁺CD57⁺ CD4⁺ and CD8⁺ T cells in control and OMS subjects. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05, **p<0.01.

Figure 2

Elevated percentage of the Tfh family in the OMS group. (A) Frequencies of Tfh family (total Tfh, Tfh-Th1, Tfh-Th17 and Tfr). (B) Frequencies of γδ family (total γδ T cells, γδ1 T cells and γδ2 T cells). (C) Frequencies of MAIT cells (total MAIT, CD4⁺ MAIT cells and CD8⁺ MAIT cells) in control and OMS subjects. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Increased frequencies of specific memory B cell subsets, plasmablasts, and Bregs in OMS subjects. Depicted are B cell family subsets in control and OMS subjects. (A) Frequencies of total B cells. (B) Frequencies of B cell subsets: naive B cells, activated memory, classical memory, atypical memory, transitional, and mature B cells. (C) Frequencies of mature B cell subsets: IgD⁺CD27⁺, IgD only memory cells, marginal zone B cells, and IgD^- B cells. (D) Frequencies of total memory B cells and plasmablasts. (E) Frequencies of memory B cell subsets (IgG⁺, IgM⁺, IgA⁺). (F) Frequencies of the Bregs subsets CD24^highCD27⁺ and CD24^highCD38^high. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05, **p<0.01.

Figure 4

Unaltered levels of myeloid compartment. (A) Frequencies of the monocyte family (classical, intermediate, and non-classical monocytes). (B) Frequencies of DC subsets (mDC and pDC). (C) Frequencies of mDC subsets (cDC1 and cDC2). (D) Frequencies of MDSC and eMDSC in control and OMS subjects. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test.

Figure 5

Increased frequencies of early NK cells. Frequencies of the NK cell family (early, mature, and terminal NK cells). The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05.

Figure 6

OMS decreases TIM3 expression on Tfh cells. MFI values of PD1 and CD57 (A) in CD4⁺ T cells and (B) in CD8⁺ T cells. MFI values of PD1 and TIM3 (C) in Tfh, (D) in γδ T cells, and in (E) MAIT cells. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05.

Figure 7

OMS infection increases the TIM3 expression on B cells. (A) MFI values of HLA-DR on B cell subsets (total, mature, naive and, memory B cells), (B) MFI values of TIM3 on B cell subsets. The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05.

Figure 8

OMS infection increases exhaustion molecule expression on the NK cell family. MFI value of (A) TIM3, (B) PDL1, (C) VISTA, and (D) CD86 in NK cell subsets (early, mature, and late). The bars represent the mean ± SD. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05, **p<0.01.

Figure 9

OMS infection increases exhaustion molecule expression on monocyte subsets. MFI value of (A) TIM3, (B) PDL1, (C) Gal-9, (D) VISTA, (E) HLA-DR, and (F) CD86 in monocyte subsets (classical, intermediate, and non-classical monocytes). Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05.

Figure 10

OMS infection increases exhaustion molecule expression on mDC and MDSC. MFI value of (A) TIM 3, (B) PDL1, (C) VISTA, (D) CD80, and (E) CD86 in mDC and MDSC. Statistical significance was determined by student t or Mann-Whitney U test and *p<0.05, ***p<0.001.