The chemokine CXCL1/KC abrogates experimental autoimmune myocarditis in mice.

<p>(A) Severe inflammation in the myocardium was accompanied by persistent presence of the chemokine MIP1α. (B) CXCL1/KC was significantly elevated in the serum of wild type BALB/c mice and significantly reduced in the serum of <i>LPS</i>^def mice. Serum chemokine concentrations were determined 21 days after the initial immunization with heart-specific M7Aα peptide in CFA (5 mice per group, Mean value ± SD). (C) Therapeutic administration of mouse recombinant chemokine CXCL1/KC, after experimental challenge with heart-specific M7Aα peptide in CFA, abrogated heart inflammatory infiltrate and heart muscle damage. Squares represent individual mice, lines indicate mean values. (D) Bland histopathologic picture in a representative heart section from a mouse treated with mouse recombinant chemokine CXCL1/KC after challenge with M7Aα peptide in CFA. Original magnifications x10 is shown. (E) Severe myocarditis in a heart section from a control mouse injected with saline after challenge with M7Aα peptide in CFA. Original magnifications x30 is shown Hearts were analyzed 21 days after the initial immunization with heart-specific M7Aα peptide in CFA. Staining was with hematoxylin and eosin (H&E). Mice were immunized with heart-specific M7Aα peptide in CFA and heart histopathology was determined at day 21 after the initial immunization. One result representative of 5 independent experiments is shown. (F) Cartoon outlining the mode of action by systemic LPS and CXCL1/KC in EAM. M7Aα peptide and CFA co-localize within the phagosme where M7Aα peptide is processed for presentation in conjunction with MHC class II molecules. TLR4 is dispensable for auto-antigen processing and presentation, TLR4, in fact, inhibits this process which, in turn, limits the autoantigen dependent expansion of pathogenetic T_effs. TLR4 promotes the production of CXCL1/KC in EAM; and CXCL1/KC is cardioprotective as exemplified by its prevention of iNOS protein induction. T, effector T lymphocyte; DC, antigen presenting dendritic cell; CM cardiomyocyte.</p

TLR4 signaling is crucial for prevalence and severity of experimental autoimmune myocarditis.

<p>(A) Systemic administration of low dose LPS curbed the induction of experimental autoimmune heart disease. Wild type BALB/c mice treated with low doses LPS developed myocarditis with significantly reduced severity and prevalence compared to saline treated controls. Mice were immunized with M7Aα peptide in CFA and treated by intraperitoneal injection 3 times, on days 5, 9, and 13 after the initial immunization, with the indicated doses of highly pure LPS or saline. Squares represent individual mice, lines indicate mean values. * p<0.05 when compared to other groups using ANOVA for multiple-sample comparisons (Bonferroni). (B) Antagonizing the LPS receptor TLR4 significantly increased severity of autoimmune myocarditis. mkLPS, which lacks the myristoyl fatty acid moiety of lipid A, antagonized TLR4 signaling through direct interaction with TLR4. Mice immunized with M7Aα peptide in CFA and mkLPS developed significantly more severe myocarditis than control mice receiving M7Aα peptide in CFA and saline. Disease severity was determined by histopathology 21 days after the initial immunization. Squares represent individual mice, lines indicate mean values. * p<0.05 by Student;s t-test. One representative result out of 4 independent experiments is shown.</p

<i>LPS</i>^def mice are highly susceptible to induction of autoimmune inflammatory heart disease.

<p>Genetic loss of TLR4 function leads to severe autoimmune myocarditis in mice challenged with heart-specific autoantigen and complete Freund's adjuvant (CFA), a complex mixture of TLR agonists. (A) <i>LPS</i>^def. mice lacking functional TLR4 developed significantly more severe autoimmune myocarditis that wild type BALB/c control mice. Histopathological disease severity, as described in Methods, was determined 21 days after the initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. One representative result out of 5 independent experiments is shown. (B) Serum IgG autoantibodies reactive to heart specific epitope M7Aα were determined 21 days after initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. One representative result out of 5 independent experiments is shown. (C) Heart inflammatory infiltrate. CD3ε⁺ T cells expressing IL-17A were evaluated 21 days after initial immunization with heart-specific M7Aα peptide and CFA. Squares represent the percentage of IL-17A⁺ cells per CD3ε⁺ T cells as determined by immunohistochemistry in heart-sections from individual mice, lines indicate mean values. * p<0.05. One representative result out of 5 independent experiments is shown. (D) Representative photomicrograph of heart section from a <i>LPS</i>^def mouse immunized with autoantigen and CFA. Inflammatory infiltrate consisting mostly of mononuclear cells is present throughout the myocardium often surrounding necrotic cardiomyocytes (arrow). Original magnifications x10 and x200 are shown. Hearts were analyzed 21 days after initial immunization with heart-specific M7Aα peptide in CFA. Staining was with hematoxylin and eosin (H&E). (E) Histopatholgy in a heart section from a BALB/c mouse immunized with heart-specific M7Aα peptide in CFA. Inflammatory infiltrate consisting mostly of mononuclear cells is present as an inflammatory focus (arrow). Original magnifications x100 is shown. (F) <i>LPS</i>^def. mice fail to resolve autoimmune myocarditis by day 28 after the initial autoantigen challenge but not wild type BALB/c control mice. Histopathological disease severity, as described in Methods, was determined 28 days after the initial immunization with heart-specific M7Aα peptide and CFA. * p<0.05. Squares represent individual mice, lines indicate mean values. One representative result out of 3 independent experiments is shown. Student's t Test was used for statistical analysis.</p

CXCL1/KC prevents the induction of iNOS protein expression in hearts of mice challenged with cardiac-specific α-myosin-heavy chain-derived peptide M7Aα.

<p>Frequency of positive cells in heart sections from mice immunized with M7Aα in CFA 21 days after the initial immunization. Percentage of cells (±SD) staining for iNOS in cardiomyocytes, or CD68 and iNOS in inflammatory cells, was calculated after counting cells per visual field at magnification of x160 on sections from hearts of 3 mice per group. A minimum 100 cells in at east 10 different visual fields was evaluated per heart. One result representative of 3 independent experiments is shown. * p<0.05 when compared to the other groups. n.d., cells were not detected in sufficient numbers, i.e., there were <100 cells per visual field.</p

LPS-induced TLR4 signaling inhibits T and B effector lymphocyte function in experimental autoimmune heart disease.

<p>(A) <i>LPS</i>^def mice have similar numbers of TCRβ⁺CD4⁺Foxp3⁺ T_regs after challenge with M7Aα peptide in CFA. The number of effector CD4⁺Foxp3⁻ T_effs was significantly increased in spleens of <i>LPS</i>^def mice after autoantigen challenge. Squares represent individual mice, lines indicate mean values. (B) LPS treatment <i>in vivo</i> dampened the proliferation of heart Ag-specific effector T cells <i>ex vivo</i>. An equal number of T_effs, isolated from spleens, either of mice immunization with M7Aα in CFA and treated either with three injections of either LPS (5 µg/kg) or from saline treated controls, was cultured with γ-irradiated syngeneic stimulator splenocytes pulsed with the M7Aα peptide. Squares represent the number of cultured cells obtained from individual mice, lines indicate mean values. (C) Suppression of proliferation of T cells (5x10⁴) from naïve mice stimulated with anti-CD3 ε (1 µg/ml) plus anti-CD28 (0.1 µg/ml) antibodies in the presence of pre-cultered T cells (1x10⁵, derived from mice immunized with M7Aα in CFA and treated either with three injections of either LPS (5 µg/kg) or saline. * p<0.05 by Student;s t-test. A representative result from 3 experiments is shown. (D) Systemic administration of low doses of LPS significantly reduced the concentration of IgG reactive to heart-specific epitope M7Aα. Mice were immunized and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089647#pone-0089647-g002" target="_blank">Figure 2</a>. * p<0.05 when compared to other groups using ANOVA for multiple-sample comparisons (Bonferroni. One representative result out of 4 independent experiments is shown.</p

Efficacy and Safety of Reparixin in Patients with Severe COVID-19 Pneumonia: A Phase 3, Randomized, Double-Blind Placebo-Controlled Study

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