Heterogeneity in FoxP3- and GARP/LAP-Expressing T Regulatory Cells in an HLA Class II Transgenic Murine Model of Necrotizing Soft Tissue Infections by Group A Streptococcus

Abstract

Invasive group A streptococcus (GAS) infections include necrotizing soft tissue infections (NSTI) and streptococcal toxic shock syndrome (STSS). We have previously shown that host HLA class II allelic variations determine the risk for necrotizing fasciitis (NF), a dominant subgroup of NSTI, and STSS by modulating responses to GAS superantigens (SAgs). SAgs are pivotal mediators of uncontrolled T-cell activation, triggering a proinflammatory cytokine storm in the host. FoxP3-expressing CD4 CD25 T regulatory cells (Tregs) comprise phenotypically and functionally heterogeneous subsets with a profound ability to suppress inflammatory responses. Specifically, activated Tregs, which express glycoprotein A repetitions predominant (GARP) and display latent transforming growth factor β1 (TGF-β1) complexes (latency-associated peptide [LAP]), exhibit strong immunosuppressive functions. The significance of Tregs that may participate in suppressing inflammatory responses during NSTI is unknown. Here, we phenotypically characterized FoxP3/GARP/LAP-expressing Tregs in GAS-infected or SAg (SmeZ)-stimulated splenocytes from transgenic (tg) mice expressing human HLA-II DRB1*15 (DR15 allele associated with nonsevere NF/STSS-protective responses) or DRB1*0402/DQB1*0302 (DR4/DQ8 alleles associated with neutral risk for combined NF/STSS). We demonstrated both and that the neutral-risk allele upregulates expression of CD4 CD25 activated effector T cells, with a significantly lower frequency of Foxp3/GARP LAP but higher frequency of Foxp3 LAP Tregs than seen with the protective allele. Additional studies revealed that the presentation of SmeZ by the neutral-risk allele significantly increases proliferation and expression of effector cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) and upregulates CD4 CD25 T cell receptors (TCRs) carrying specific Vβ 11 chain (TCRVβ11) T cells and Th1 transcription factor mRNA levels. Our data suggest that neutral-risk alleles may drive Th1 differentiation while attenuating the induction of Tregs associated with suppressive function.

HLA-II-dependent neuroimmune changes in Group A Streptococcal Necrotizing Fasciitis

“…Therefore, one future direction is to evaluate the neuroinflammation potential of several clinical strains of GAS across a battery of HLA-II transgenic mice. We considered HLA-II transgenic mice as a clinically relevant and translational model to study the neuroinflammation effects of peripheral inflammation involved in GAS infections since these mice mimic responses seen in humans [29,57]. Notwithstanding that the magnitude of responses is directly linked to HLA-II allelic variations, it is understandable that a single gene cannot hold the key to all the intricacies of disease pathogenesis underlying GAS infections.…”

“…The spleen, brain, and necrotic skin were recovered from each mouse under sterile conditions. The necrotic skin was homogenized using a motorized homogenizer (Omni International, Marietta, GA), and the GAS burden was enumerated by preparing tenfold dilutions in DPBS and plated on sheep blood agar as described previously [29,36]. The brains were hemisected, and the hippocampus was isolated from the right hemisphere.…”

HLA-II Alleles Influence Physical and Behavioral Responses to a Whey Allergen in a Transgenic Mouse Model of Cow's Milk Allergy

“…Male and female transgenic mice expressing HLA-II alleles, DRB1 * 03:01 (DR3), DRB1 * 15:01 (DR15), or HLA-DQA1 * 0301 and DQB1 * 0302 (DQ8), were bred in-house in a specific pathogen-free room at the University of North Dakota. These transgenic mice have been shown to express functional HLA ( 32 , 33 ), and the presence of the appropriate HLA-II transgenes was confirmed by PCR-based genotyping using allele-specific oligonucleotide primers as described previously ( 33 , 34 ). Animals were housed under a 12-h light/12-h dark cycle and had ad libitum access to water and a whey-protein-free diet (Teklad 2018, Envigo Corporation, Indianapolis, IN, USA).…”

Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

“…A representative M1T1 clonal Group-A-Streptococcus GAS5448 was used for subcutaneous infections ( 91 ). GAS was grown at 37°C under static conditions in Todd-Hewitt broth (BD, MD, USA) supplemented with 1.5% yeast extract (BD Biosciences, MD, USA) and in vivo infections were performed as described previously ( 29 , 92 ). WT and Mt3 -/- mice (n=8/group) were used in this study as a model for subcutaneous GAS infections.…”