The Critical Role of Chemokine (C–C Motif) Receptor 2-Positive Monocytes in Autoimmune Cholangitis

The therapy of primary biliary cholangitis (PBC) has lagged behind other autoimmune diseases despite significant improvements in our understanding of both immunological and molecular events that lead to loss of tolerance to the E2 component of pyruvate dehydrogenase, the immunodominant autoepitope of PBC. It is well known that Ly6Chi monocytes are innate immune cells infiltrating inflammatory sites that are dependent on the expression of C–C motif chemokine receptor 2 (CCR2) for emigration from bone marrow. Importantly, humans with PBC have a circulating monocyte pro-inflammatory phenotype with macrophage accumulation in portal tracts. We have taken advantage of an inducible chemical xenobiotic model of PBC and recapitulated the massive infiltration of monocytes to portal areas. To determine the clinical significance, we immunized both CCR2-deficient mice and controls with 2OA-BSA and noted that CCR2 deficiency is protective for the development of autoimmune cholangitis. Importantly, because of the therapeutic potential, we focused on inhibiting monocyte infiltration through the use of cenicriviroc (CVC), a dual chemokine receptor CCR2/CCR5 antagonist shown to be safe in human trials. Importantly, treatment with CVC resulted in amelioration of all aspects of disease severity including serum total bile acids, histological severity score, and fibrosis stage. In conclusion, our results indicate a major role for Ly6Chi monocytes and for CCR2 in PBC pathogenesis and suggest that inhibition of this axis by CVC should be explored in humans through the use of clinical trials

Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-), FoxP3(-)) and suppressor (CD25(+), FoxP3(+)) CD4(+) T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4(+) subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+)CD25(-) T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis

Apoptosis of Purified CD4+ T Cell Subsets Is Dominated by Cytokine Deprivation and Absence of Other Cells in New Onset Diabetic NOD Mice

BACKGROUND: Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures. PRINCIPAL FINDINGS: Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25(+) T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25(-) T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25(+) T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays. CONCLUSIONS: The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression

Soluble and cellular factors affecting susceptibility to apoptosis.

<p><b>A</b>. CD25⁻ and CD25⁺ T cells isolated from new onset diabetic NOD females were incubated for 48 hours in conditioned medium from CD25⁻ T cells stimulated with surface-bound anti CD3 and anti-CD28 antibodies (n = 3). Data are compared to corresponding measurements of isolated cells and mixed cultures. <b>B</b>. Spontaneous apoptosis after 48 hours of culture of isolated CD25⁺ T cells (n = 4) and gated subsets in mixed cultures (n = 5) following B220, GR-1 and MAC-1 depletion (n = 4). <b>C</b>. Equal numbers of isolated CD25⁻ and CD25⁺ T cells from diabetic NOD mice were mixed for determination of apoptosis after 48 hours of culture in the CD25+ subset (gate). Data are representative of four independent incubations.</p

Influence of IL-2 on CD4⁺ T cell apoptosis.

<p><b>A</b>. Apoptosis of CD4⁺CD25⁻ (n = 5) and CD4⁺CD25⁺ T cells (n = 4) isolated from new onset diabetic NOD females and incubated for 48 hours with and without 2000 U/ml IL-2. <b>B</b>. Demonstrative measurements of proliferation rates of isolated CD25⁻ and CD25⁺ cells as determined from CFSE dilution (representative of 4 independent measurements). <b>C</b>. Impact of exogenous IL-2 supplementation on apoptosis measured by gating on CD25⁻ and CD25⁺ subsets in mixed cultures (n = 4). <b>D</b>. Demonstrative plots of CFSE dilution in gated CD4⁺ subsets within mixed cultures (representative of 4 measurements).</p

Sensitivity to apoptosis of isolated CD4⁺ T cells under CD3 and CD28 stimulation.

<p>CD25⁻ and CD25⁺ T cells isolated from new onset diabetic NOD mice were stimulated with beads conjugated to anti-CD3 and anti-CD28. <b>A</b>. Apoptosis during 48 hours of incubation under CD3 (n = 4) and CD3/CD28 stimulation (n = 4). <b>B</b>. Proliferation rates determined from CFSE dilution. <b>C</b>. Isolated CD25⁻ T cells convert to express CD25 without FoxP3 priming during 48 hours of CD3/CD28 stimulation. Isolated CD25+ T cells sustain Cd25 and FoxP3 expression during Cd3/Cd28 stimulation. <b>D</b>. Proliferation rates of cells unresponsive to stimulation (CD25⁻) and the subset that upregulates CD25 expression (CD25⁻→CD25⁺) and representative plots of CFSE dilution. <b>E</b>. Fractional apoptosis of responsive and unresponsive CD25⁻ T cells under CD3/CD28 stimulation (n = 4) and representative plots of apoptosis.</p

Apoptosis under CD3/CD28 stimulation.

<p><b>A</b>. Representative plots demonstrating upregulation of CD25 in response to CD3/CD28 stimulation in mixed splenocyte cultures. <b>B</b>. Spontaneous apoptosis in isolated (n = 4) and gated cells (n = 5) in reference to responsiveness (CD25 expression) to CD3-activation, assuming negligible contribution of naturally occurring CD25⁺ T cells in mixed cultures. <b>C</b>. Proliferation rates under CD3-activation of responsive (CD25⁻→CD25⁺) and non-responsive (CD25⁻) subsets in isolated CD25⁻ T cells (n = 4) and in mixed cultures (gated, n = 5). <b>D</b>. Fractional apoptosis of responsive and non-responsive cells to CD3/CD28 stimulation in mixed cultures (n = 5). <b>E</b>. Proliferation rates under CD3/CD28 stimulation.</p