Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

Interleukin-10 (IL-10) is known to inhibit IL-12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor-kB (NF-kB). We demonstrate that the c-rel transcription factor also plays a major role in IL-10-mediated IL-12 suppression. Treatment of macrophages with recombinant IL-10 inhibited nuclear c-rel levels, whereas addition of neutralizing anti-IL-10 antibody up-regulated both nuclear c-rel levels and IL-12 production by macrophages. Decreased nuclear c-rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IkBα ) in the cytoplasm, indicating that IL-10 prevents degradation of IkBα and the subsequent translocation of c-rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c-rel at a concentration of 10 nm, when used with anti-IL-10 antibody, resulted in reduced expression of IL-12. In a complementary experiment, in vitro transient expression of p65 NF-kB could not rescue the inhibitory effect of IL-10 on IL-12 production, suggesting that NF-kB alone was not sufficient to restore IL-12 production during IL-10 treatment. However, over-expression of c-rel resulted in IL-12 restoration upon stimulation with lipopolysaccharide plus interferon-γ - during IL-10 treatment. Our studies highlight the involvement of c-rel in IL-10-mediated IL-12 regulation

The Co-Operonic PE25/PPE41 Protein Complex of Mycobacterium tuberculosis Elicits Increased Humoral and Cell Mediated Immune Response

BACKGROUND: Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. METHODOLOGY/PRINCIPAL FINDING: We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15-22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8-10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-gamma, TNF-alpha and IL-2 cytokines. CONCLUSION: While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis

PPE Antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein

The cause–effect relation of tuberculosis on incidence of diabetes mellitus

Tuberculosis (TB) is one of the oldest human diseases and is one of the major causes of mortality and morbidity across the Globe. Mycobacterium tuberculosis (Mtb), the causal agent of TB is one of the most successful pathogens known to mankind. Malnutrition, smoking, co-infection with other pathogens like human immunodeficiency virus (HIV), or conditions like diabetes further aggravate the tuberculosis pathogenesis. The association between type 2 diabetes mellitus (DM) and tuberculosis is well known and the immune-metabolic changes during diabetes are known to cause increased susceptibility to tuberculosis. Many epidemiological studies suggest the occurrence of hyperglycemia during active TB leading to impaired glucose tolerance and insulin resistance. However, the mechanisms underlying these effects is not well understood. In this review, we have described possible causal factors like inflammation, host metabolic changes triggered by tuberculosis that could contribute to the development of insulin resistance and type 2 diabetes. We have also discussed therapeutic management of type 2 diabetes during TB, which may help in designing future strategies to cope with TB-DM cases

The evil axis of obesity, inflammation and Type-2 Diabetes

Obesity and Type 2 Diabetes (T2D) are global problems affecting all age groups and have been characterized as lifestyle disorders. Though no study has clearly proved a direct correlation between obesity and T2D, a number of factors are associated with obesity causing insulin resistance and T2D. The factors such as adipokines and various transcription factors help to maintain a proper metabolic state in the body. Deregulation in any of these signaling balances due to obesity may trigger an inflammatory cascade which could lead to the aforesaid problems of insulin resistance and T2D. In this review, we have discussed the factors that probably link inflammation to obesity-induced insulin resistance and subsequently T2D and the possible therapeutic opportunities to decrease health risk of T2D in future

Therapeutic application of PPE2 protein of Mycobacterium tuberculosis in inhibiting tissue inflammation

Abstract There is an increasing need to develop biological anti‐inflammatory agents that are more targeted, effective, and with lesser side effects as compared to conventional chemical drugs. In the present study, we found that Mycobacterium tuberculosis protein PPE2 and a synthetic derivative peptide can suppress the mast cell population and inhibit several vasoactive and fibrogenic mediators and pro‐inflammatory cytokines induced by mast cells in formalin‐induced tissue injury. PPE2 was found to inhibit transcription from the promoter of stem cell factor, important for mast cell maintenance and migration. Thus, PPE2/peptide can be used as a potent nonsteroidal therapeutic agent for the treatment of inflammation and tissue injury

The PPE18 protein of Mycobacterium tuberculosis Inhibits NF-&#954; B/rel-mediated proinflammatory cytokine production by upregulating and phosphorylating suppressor of cytokine signaling 3 protein

Mycobacterium tuberculosis bacteria are known to suppress proinflammatory cytokines like IL-12 and TNF-&#945; for a biased Th2 response that favors a successful infection and its subsequent intracellular survival. However, the signaling pathways targeted by the bacilli to inhibit production of these cytokines are not fully understood. In this study, we demonstrate that the PPE18 protein of M. tuberculosis inhibits LPS-induced IL-12 and TNF-&#945; production by blocking nuclear translocation of p50, p65 NF-&#954;B, and c-rel transcription factors. We found that PPE18 upregulates the expression as well as tyrosine phosphorylation of suppressor of cytokine signaling 3 (SOCS3) and the phosphorylated SOCS3 physically interacts with I&#954;B&#945;–NF-&#954;B/rel complex, inhibiting phosphorylation of I&#954;B&#945; at the serine 32/36 residues by I&#954;B kinase-&#946; and thereby prevents nuclear translocation of the NF-&#954;B/rel subunits in LPS-activated macrophages. Specific knockdown of SOCS3 by small interfering RNA enhanced I&#954;B&#945; phosphorylation, leading to increased nuclear levels of NF-&#954;B/rel transcription factors vis-a-vis IL-12 p40 and TNF-&#945; production in macrophages cotreated with PPE18 and LPS. The PPE18 protein did not affect the I&#954;B kinase-&#946; activity. Our study describes a novel mechanism by which phosphorylated SOCS3 inhibits NF-&#954;B activation by masking the phosphorylation site of I&#954;B&#945;. Also, this study highlights the possible mechanisms by which the M. tuberculosis suppresses production of proinflammatory cytokines using PPE18. The macrophages elicit various antimycobacterial mechanisms during the innate phase of activation that play crucial roles in deciding the outcome of Mycobacterium tuberculosis infection (1). Among these, production of proinflammatory cytokines like IL-12 and TNF-&#945; is critical for mounting an optimal defense against mycobacterial infection (2–5). The TNF-&#945; is important for granuloma formation and induction of cytotoxicity against the bacilli (6, 7). In contrast, IL-12 plays a crucial role in activating the protective Th1 immune response (5, 8, 9). Regulation of proinflammatory responses often involves diverse signaling cascades, and the pathogenic M. tuberculosis bacteria have evolved several mechanisms to modulate these signaling pathways to suppress IL-12 and TNF-&#945; production to favor its long-term survival and persistence inside the host (10–12). Interestingly, virulence of pathogenic mycobacteria is found to be inversely correlated with the levels of IL-12 and TNF-&#945; secreted (11–14). Human Monocyte-Derived Macrophages (MDMs) are shown to produce a significantly lower amount of IL-12 after exposure to M. tuberculosis than other bacteria (9), indicating that M. tuberculosis specifically targets the signaling cascades that lead to production of this cytokine. However, the molecular mechanisms by which the pathogenic mycobacteria can downregulate induction of these proinflammatory cytokines are not fully understood. The mycobacteria are characterized by the presence of two unique gene families, the proline-glutamic acid and the proline-proline-glutamic acid (PPE) (15), which are highly expanded in pathogenic species like M. tuberculosis (16). Although the proteins are predicted to play a role in generating antigenic variation (17–20), the functional role of these proteins in mycobacterial pathogenesis is not clearly understood. Some of these genes are predicted to play an important role in mycobacterial persistence inside the host and are crucial in M. tuberculosis pathogenesis (21–23). Recently, we reported that a PPE family of proteins of M. tuberculosis, PPE18, inhibits the Th1-type response by specifically downregulating IL-12 p40 and other proinflammatory indicators (24). We found that PPE18 binds to the TLR2 leucine-rich repeat (LRR) 11–15 region and causes an inhibition of IL-12 p40 (24). It appears that PPE18 probably targets the proinflammatory signaling downstream of TLR2 by activating certain negative regulators of this pathway. Interestingly, suppressor of cytokine signaling 3 (SOCS3) downstream of TLR2 (25) acts as negative regulator of proinflammatory signaling (26–29). Because several pathogenic mycobacterial species induce expression of the SOCS3 protein (30–32), we speculated that probably SOCS3 protein is involved in the suppression of induction of IL-12 p40 in macrophages treated with PPE18. The SOCS3 protein is known to play important roles in the regulation of proinflammatory responses during infection (25–28, 33, 34). Constitutively expressed SOCS3 was found to favor development of Th2-type response and correlate well with the severity of Th2-mediated disease like atopic asthma (35). Further, SOCS3-transduced dendritic cells are found to be effective inducers of Th2-type T cells in vitro and in vivo through reduced production of IL-12, IFN-&#947; and IL-23 p19 cytokines (36). Although the mechanisms are not clear, SOCS3-transduced dendritic cells are shown to be effective inducers of Th2-type T cells in vitro and in vivo (36). Various studies have indicated a direct role of SOCS3 in downregulating the IFN-&#947; signaling pathway (37, 38). SOCS3 is known to predominantly target the STAT family of proteins to attenuate IFN-&#947;–specific proinflammatory signaling (31, 39, 40). But it could be possible that the effect of SOCS3 is not restricted to STAT signaling alone but also to other signaling pathways to downregulate IL-12 and TNF-&#945; cytokines during tuberculosis. Although mycobacteria are known to increase SOCS3 expression in macrophages (31, 32), their significance in the macrophage innate-effector response in tuberculosis is not well understood. It is possible that the bacilli exploit the SOCS3 signaling pathway to inhibit proinflammatory cytokine induction in macrophages. In this study, we describe a unique signaling pathway responsible for the SOCS3-mediated downregulation of proinflammatory cytokines that involves the I&#954;B&#945;–NF-&#954;B/rel signaling and conclude that PPE18 targets this signaling pathway to suppress induction of IL-12 p40 and TNF-&#945; cytokines in activated macrophages to favor a Th2-type response (24) that helps its replication and persistence of the bacilli inside the hosts