Mesenchymal stem cell-conditioned medium reduces disease severity and immune responses in inflammatory arthritis

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis

Modification of the carboxy-terminal flanking region of a universal influenza epitope alters CD4+ T-cell repertoire selection

Human CD4+ αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA305−320) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4+ T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection

A Mycobacterium leprae Hsp65 Mutant as a Candidate for Mitigating Lupus Aggravation in Mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K409A pep synthetic peptides, which cover residues 352–371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K409A+Leader pep or K409A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352–371 region. The number of interactions observed for WT is much higher than for Hsp65 K409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F1 mice were inoculated with Hsp60 or K409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases

A case of mistaken identity: HSPs are no DAMPs but DAMPERs

Until recently, the immune system was seen solely as a defense system with its primary task being the elimination of unwanted microbial invaders. Currently, however, the functional significance of the immune system has obtained a much wider perspective, to include among others the maintenance and restoration of homeostasis following tissue damage. In this latter aspect, there is a growing interest in the identification of molecules involved, such as the so-called danger or damage-associated molecular patterns (DAMPs), also called alarmins. Since heat shock proteins are archetypical molecules produced under stressful conditions, such as tissue damage or inflammation, they are frequently mentioned as prime examples of DAMPs (Bianchi, J Leukoc Biol 81:1–5, 2007; Kono and Rock, Nat Rev Immunol 8:279–289, 2008; Martin-Murphy et al., Toxicol Lett 192:387–394, 2010). See for instance also a recent review (Chen and Nunez, Science 298:1395–1401, 2010). Contrary to this description, we recently presented some of the arguments against a role of heat shock protein as DAMPs (Broere et al., Nat Rev Immunol 11:565-c1, 2011). With this perspective and reflection article, we hope to elaborate on this debate and provide additional thoughts to further ignite this discussion on this critical and evolving issue

Heat-Killed Trypanosoma cruzi Induces Acute Cardiac Damage and Polyantigenic Autoimmunity

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freund's adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation

Altered Th17/Treg balance and dysregulated IL-1β response influence susceptibility/resistance to experimental autoimmune arthritis

This study was aimed at gaining an insight into immune mechanisms of differential susceptibility to autoimmunity of individuals sharing the same major histocompatibility complex by studying arthritis-susceptible Lewis (LEW) and arthritis-resistant Wistar Kyoto (WKY) rats (both RT.1l) using the adjuvant arthritis (AA) model of rheumatoid arthritis (RA). Lymph node cells (LNC) and synovium-infiltrating cells (SIC) of LEW and WKY rat subjected to an arthritogenic challenge were tested. The frequency of T helper 17 (Th17) and T regulatory (Treg) cells was determined by flow cytometry, whereas serum and spleen adherent cell (SAC)-derived supernatant were analyzed for specific cytokines and chemokines. We observed that WKY rats are not deficient in generating a Th17 response to the arthritogenic challenge in LNC (periphery); however, the Th17/Treg ratio is markedly reduced in the joint (target organ) of WKY versus LEW rats because of reduced Th17 levels therein in WKY rats. These results suggest differential and selective decrease in Th17 cell migration into the joints of WKY rats. Interestingly, serum levels of chemokines RANTES and MCP-1 were reduced in WKY rats. Furthermore, WKY rats showed reduced serum IL-1β level in vivo but no defect in IL-1β production by SAC in vitro, suggesting an effective in vivo regulation of IL-1β response. We also unraveled the role of interferon-γ (IFNγ), which we have previously reported to be increased in WKY versus LEW rats, in regulation of IL-1β. Thus, reduced Th17/Treg ratio in the target organ (joints) and decreased systemic IL-1β might contribute to the AA-resistance of WKY rats; whereas the converse factors render LEW more vulnerable to AA. </jats:p

Huo-Luo-Xiao-Ling Dan modulates antigen-directed immune response in adjuvant-induced inflammation

Ethnopharmacological relevance: HLXL is a traditional Chinese medicine that has long been used in folk medicine for the treatment of chronic inflammatory diseases. However, the precise immunological mechanisms by which HLXL mediates its anti-inflammatory activity are not fully defined. Aim of the study: To determine the effects of HLXL on antigen-specific immune parameters in adjuvant-induced inflammation model in the Lewis rat. Materials and methods: Rats were fed daily with either HLXL (2.3 g/kg) or vehicle (water) beginning 3 days before subcutaneous injection of heat-killed Mycobacterium tuberculosis H37Ra (Mtb), and then continued for another 6 days. After 9 days of Mtb injection, the draining lymph node cells were tested for T cell proliferative and cytokine responses against mycobacterial heat-shock protein 65 (Bhsp65). Moreover, sera were tested for anti-Bhsp65 antibodies and nitric oxide (NO). Results: HLXL-treated rats showed reduced T cell proliferative response to Bhsp65 compared to control rats. Furthermore, HLXL suppressed IL-17 response but enhanced IL-10 response without much effect on IFN-γ. HLXL treatment also reduced the levels of anti-Bhsp65 antibodies but not that of NO. Conclusions: HLXL feeding modulated both the cellular and the humoral immune response to Bhsp65 favoring an anti-inflammatory milieu for the suppression of adjuvant-induced inflammation. © 2009 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex

Extract of the Chinese herbal formula Huo Luo Xiao Ling Dan inhibited adjuvant arthritis in rats

Ethnopharmacological relevance: The herbal formula Huo Luo Xiao Ling Dan (HLXL) and its modifications have been used in traditional Chinese medicine for about one hundred years to alleviate pain and inflammation. Aim: To investigate the effects of HLXL on complete Freund's adjuvant (CFA)-induced multiple-joint arthritis in rats. Materials and methods: Male Lewis rats, 190-210 g, were immunized subcutaneously at the base of the tail with 200 μl of heat-killed Mycobacterium tuberculosis in mineral oil (5 mg/ml). HLXL (2.30 and 4.60 g/kg) or vehicle control (n = 8 per group) was administered orally (i.g.) once a day between days 16 and 25 post-CFA injection. The rats were observed for signs of arthritis with arthritic changes (erythema, edema, induration) being scored on a scale of 0-4 of increasing severity using a standard scoring system. The maximum arthritis score per rat was 16. A plethysmometer was used to measure edema volume in each paw. Adverse effects of HLXL were monitored by closely observing the animals for unusual behavioral changes. Levels of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in local tissue were measured by enzyme-linked immunosorbent assay on day 25 post-CFA. Results: HLXL significantly decreased arthritis scores between days 23-25 in the 2.30 g/kg group and 21-25 in the 4.60 g/kg group (p < 0.05). It reduced paw edema on days 22 and 24 in the 2.30 g/kg group and on days 20, 22 and 24 in the 4.60 g/kg group compared to control (p < 0.05). Local tissue TNF-α and IL-1β levels on day 25 post-CFA injection were significantly (p < 0.05) lower in rats treated with HLXL than in control rats. No observable adverse effects were found. Conclusion: The data suggest that HLXL produces significant anti-arthritic effects that may be mediated by suppressing pro-inflammatory cytokines, and it appears to be safe. © 2008 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex