Ratna Biswas

Prolactin regulates antitumor immune response through induction of tumoricidal macrophages and release of IL-12

The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL‐Rs in these cells. Here, we show that PRL, though it failed to activate mouse peritoneal resident macrophages (RMs), acted as a second signal and activated mouse peritoneal inflammatory macrophages (EMs) to a tumoricidal state. The cytotoxicity of mouse tumor‐associated macrophages (TAMs) isolated at day 1 of tumor (Ehrlich ascites carcinoma, EAC) growth was enhanced by PRL. However, with progression of tumor growth, TAMs became nonresponsive to the hormone. PRL‐induced killing of P815 target cells by EMs and TAMs was independent of TNF but correlated with the hormone‐induced augmentation of NO2− and O2− release in these macrophages. Administration of PRL in vivo inhibited EAC growth and augmented NO2− release by TAMs. PRL synergized with the TH1 cytokine IFN‐γ, a known activator of macrophages, in inducing tumor killing and release of NO2− from EMs and TAMs. The hormone might activate macrophages at least partially, through the release of IFN‐γ as anti‐IFN‐γ blocked IFN‐γ– as well as PRL‐induced cytotoxicity in EMs. The TH2 cytokine IL‐4 suppressed PRL‐induced activation of macrophages. PRL induced release of IL‐12 from EMs also, which suggested that the hormone might drive the TH1 response through IL‐12. Our observations further suggest that PRL alone and in synergy with IFN‐γ, released through induction of IL‐12, may generate tumoricidal macrophages and thus regulate the antitumor immune response of tumor hosts.

Prolactin induced reversal of glucocorticoid mediated apoptosis of immature cortical thymocytes is abrogated by induction of tumor

Glucocorticoid (GC) and prolactin (PRL) are the two neuroendocrines that regulate thymocyte differentiation and maintain the immune homeostasis during stress. We found that dexamethasone (Dex), a synthetic GC, induced apoptosis in normal immature cortical thymocytes which remained unaltered in the presence of Elrichs ascitic carcinoma (EAC). PRL protected the normal CD4+ CD8+ cortical thymocytes from Dex-induced apoptosis but failed to alter the effect of Dex in tumor-bearing mice. Dex-treated normal thymocytes became unresponsive to PRL in presence of tumor cell culture supernatant. Low binding affinity of the microsomal membranes of thymocytes to PRL and absence of the mRNA of a particular form of prolactin receptor (PRL-R) suggest the presence of a different PRL-R in CD4+ CD8+ thymocytes of EAC-bearing mice. The induction of tumor may alter the PRL-R that can be correlated with the failure of PRL in rescuing CD4+ CD8+ immature cortical thymocytes from GC induced death.

LPS stimulates and Hsp70 down-regulates TLR4 to orchestrate differential cytokine response of culture-differentiated innate memory CD8(+) T cells.

Nonconventional innate memory CD8(+) T cells characteristically expressing CD44, CD122, eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) were derived in culture from CD4(+)CD8(+) double positive (DP) thymocytes of normal BALB/c and C57BL/6 mice. These culture-differentiated cells constitutively express toll-like receptor (TLR)4 and release interferon (IFN)-γ and interleukin (IL)-10. We show the TLR4-ligand lipopolysaccharide (LPS) stimulate the TLR and up-regulate IFN-γ skewing the cells towards type 1 polarization. In presence of LPS these cells also express suppressor of cytokine signaling (SOCS)1 and thus suppress IL-10 expression. In contrast, heat shock protein (Hsp)70 down-regulated TLR4 augmenting the anti-inflammatory cytokine IL-10. In association with IL-10 release IFN-γ was abrogated. The programmed cell death (PD)-1 mostly present in regulatory T cells was stimulated in these IL-10 producing cells by Hsp70 and not LPS indicating the cells can be driven to two contrast outcomes by the two TLR4 ligands. Our work provides a scope for in vitro monitoring of CD8(+) T cells to decipher important immune therapeutic option during infection or sepsis.

IL-10 alters prolactin receptor activity emulating that during breast cancer.

Peripheral blood mononuclear cells (PBMC) of breast cancer patients show altered prolactin (PRL)-induced proinflammatory response and express short form of prolactin receptor (PRL-R), besides secreting elevated level of interleukin (IL)-10 than that of the normal counterparts. IL-10 depleted the functional long form of PRL-R mRNA and protein, expressed PRL-R (SF) mRNA and blocked the PRL response found in normal individuals, which could be a mechanism to suppress the proinflammatory immune responses during malignancy.

Tumor associated release of interleukin-10 alters the prolactin receptor and down-regulates prolactin responsiveness of immature cortical thymocytes

The soluble factors produced either by Ehrlichs ascites carcinoma (EAC) or thymic adherent cells (TAC) of tumor-bearing mice comprising of CD11b(+) and CD11c(+) antigen-presenting cells caused a sharp decrease in prolactin (PRL)-induced ConA-mediated effect on survival of PNA(+) thymocytes. Similar suppression of PRL-induced effect was observed when the cells were cocultured with TAC of EAC-bearing mice. Anti-IL-10 antibody could reverse the PRL inability to induce ConA-mediated effect on PNA(+) thymocyte survival, indicating the presence of IL-10 in EAC culture supernatant (EAC sup) and thymic microenvironment. IL-10 could block PRL-induced proliferation of PNA(+) thymocytes without affecting spontaneous apoptosis. IL-10 altered the expression of the long-form (LF) of PRL-R and reduced the PRL binding of the cells, suggesting down-regulation of the PRL effect on PNA(+) thymocyte by the cytokine. Induction of tumor, which was found to increase the IL-10 secretion by TAC, also modified the PRL-R (LF) to PRL-R (SF). Since PRL plays a role in survival, proliferation and differentiation of lymphoid progenitor cells, the tuning of PRL action by IL-10 may be a possible mechanism of depletion of immature cortical thymocytes and thymic atrophy in tumor-bearing mice.

Prolactin regulates macrophage and NK cell mediated inflammation and cytotoxic response against tumor

Abstract Prolactin (PRL) is known to stimulate lymphocyte growth and function, but its role in regulating innate immune response is not clear. We show that PRL, as a secondary signal, can induce tumoricidal macrophages in mice and augment tumor target killing by human monocytes and NK cells. PRL also induces IL-1, NO 2 − , and O 2 − release by the macrophages. Through release of IL-12 and IFN-γ, PRL regulates activation of monocytes/macrophages and NK cells. PRL mediated factivation of monocytes/macrophages is inhibited by IL-4 and in malignancy. A defect in PRL-receptor expression by macrophages may occur in malignancy.

Porin differentiates TLR mediated proinflammatory response of follicular zone B cell from TLR-unresponsive IL-10 expressing marginal zone B cell

TLR-ligands are frequently chosen as candidates for vaccine or adjuvant development because they can primarily bridge innate signaling with adaptive immune responses. Since the adjuvant action of porin, the major outer membrane protein commonly present on Gram-negative bacteria, has been tested on several antigen-presenting cells, we investigated its role in driving systemic immunity which is considered a benchmark for a successful adjuvant. Here, we show porin differentially regulated splenic marginal zone (MZ) and follicular zone (FO) B cell responses in contrast to other classical TLR2-ligands FSL-1 and Pam3CSK4. The protein up-regulated TLR2 and TLR6 and stimulated the activation and costimulatory molecules on FO B cells skewing the cells toward TLR-dependent type-1 cytokine response. However, porin could not up-regulate the TLRs and activate MZ B cells. These cells responded to porin by expressing toll-interacting protein (TOLLIP), the TLR2 and -4 signaling inhibitor along with stimulation of the intracellular pathogen recognition receptor NLR caspase recruitment domain containing protein 5 (NLRC5). The CD1d(hi) MZ B cells released IL-10 unequivocally demonstrating regulatory B cell feature. Immunization with porin also resulted in transient IL-10 expression by the CD19(+)CD21(hi) B cells prior to plasma cell formation. Moreover, the plasma cells developed from the B-2 cell subsets show marked variation in generation of immunoglobulin subclasses. The work delineates multi-faceted role of B cell subsets induced by porin for robust immunity without compromising with the checks and controls.

Alternative TLRs are stimulated by bacterial ligand to induce TLR2-unresponsive colon cell response.

Although pathogenic bacteria penetrate colonic cells causing infection, the role of its surface molecules serving as key Toll-like receptor (TLR) ligands and triggering response remains unexplored. We show that TLR2-ligand porin up-regulated TLR4 on HT-29 cells, which the TLR4-ligand LPS could not. TLR1 that co-express with TLR2 got stimulated with TLR4. Besides the two TLRs, MD-2 was expressed revealing that the TLR4 co-receptor is not exclusive for LPS signaling. SARM-1 that mostly down-regulates TLR-signaling, demonstrated central role in signaling by engaging IRF-3 and NF-κB for cell activity. Porin induced type 1 chemokines particularly MCP-3, while porin-stimulated HT-29 culture supernatant displayed PBMC migration, collectively suggesting that the chemokines influence colon and immune cell cross-talk. In TLR2 down-regulated HT-29 cells, we found TLR1 and TLR4 as substitute TLRs to identify porin and orchestrate signaling. Thus, TLR replacement for PAMP recognition demonstrates specificity of ligand·TLR association can compromise and is a necessary alternative for successful execution of immune responses.

IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression.

Interleukin (IL)-15 is known to strongly modulate T-cell function; however, its role in controlling mucosal immunity, including its ability to modulate B-1a cell activity, remains to be elucidated. Here, we show that IL-15 upregulates activation molecules and the costimulatory molecule CD80 on viable B-1a cells. Cell activation was accompanied by the depletion of sialic acid-binding immunoglobulin-like lectin (Siglec)-G, an inhibitor of cell activation that is present on B-1a cells. The IL-15 receptor CD122 was stimulated on B-1a cells by the cytokine showing its direct involvement in IL-15-mediated responses. IL-10 is responsible for the long term survival of B-1a cells in culture, which is initially promoted by IL-15. The upregulation of IL-10 was followed by the appearance of suppressor of cytokine signaling (SOCS)1 in the presence of IL-15 and the loss of IL-10. This resulted in the cells switching to IL-12 expression. This anti-inflammatory to pro-inflammatory shift in the B-1a cell character was independent of the cell-specific marker CD5, which remained highly expressed throughout the in vitro life of the cells. The presence of the immunosuppressive receptor programmed cell death (PD)-1 and its ligand PD-L2 were features of a predominantly IL-10 response. PD-1 and PD-L2 can mediate juxtacrine signaling. However, the abrogation of PD-1 and its ligand was observed when the cells expressed IL-12. This demonstrates an inverse relationship between the receptor and ligand and the pro-inflammatory cytokine. The induction of IgM and IgA, which can play pivotal roles in mucosal immunity, was promoted in the presence of IL-15. Collectively, the data implicate IL-15 as the master cytokine that induces B-1a cells to mount a mucosal immune response.

Culture-differentiated CD8 + T cells acquire innate memory-like traits and respond to a pathogen-associated molecule

Selection of conventional CD4+ or CD8+ T cells is usually driven by the interaction of double‐positive CD4+CD8+ thymocytes with epithelial cells. Here, we demonstrate preferential selection of CD8+ thymocytes from in vitro differentiation of CD4+CD8+ double‐positive thymocytes exhibiting the characteristics of nonconventional innate memory CD8+ cells. In contrast to conventional CD8+ thymocytes, these culture‐differentiated CD8+ cells are eomesodermin positive and robustly express CXCR3, CD44, CD122 and TLR2. Interestingly, the pathogen‐associated molecule porin promotes preferential differentiation of the CD8+ single‐positive subset in association with promyelocytic leukemia zinc‐finger upregulation and interleukin (IL)‐4 production. On priming with anti‐CD3 antibody, porin augmented TLR2 and IFN‐γ indicating a role of the TLR ligand in acquisition of innate memory response of CD8+ thymocytes. In addition, porin has a cooperative role with IL‐15 on the expansion of memory‐phenotype CD8+ T cells along with its effector function. Thus, the study opens an avenue to unfold the cues for development of these cells and the strategies adopted for bolstering immunity during primary infection.