IL-15 stimulates NKG2D while promoting IgM expression of B-1a cells.

Interleukin (IL)-15, a key manipulator of T-cell function also modulates B-1a cell activity by augmenting activation markers, turning them towards type 1 polarization and immunoglobulin (Ig) expression which is significant in the context of gut immunity. Here we show, for the first time, IL-15 mediated up-regulation of the activation receptor NKG2D and its adaptor DAP10 in B-1a cells indicating their essential coupling with IL-15 receptor signaling pathway. Our results demonstrate IL-15 treatment increases phosphorylation of STAT5 and p38 leading to translocation of NF-κB onto the nucleus, an attribute that delineates activation of B-1a cells and its role in inflammation. In parallel, increase of anti-apoptotic Bcl-xL suggests its role in long term survival of B-1a cells in culture by IL-15. The cytokine induced overexpression of the plasma cell differentiation transcription factor BLIMP-1 while reducing PAX-5a that could be responsible for the spontaneous Ig secretion by B-1a cells. Up-regulation of IgM transcripts in presence of IL-15 validates mucosal response of the cells through natural Abs to counter pathogens.

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.

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.

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.

Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages.

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the ß-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

2019-2020 Bushfire impacts on sediment and contaminant transport following rainfall in the Upper Murray River catchment.

During the 2019-2020 Australian bushfire season, large expanses (~47%) of agricultural and forested land in the Upper Murray River catchment of southeastern (SE) Australia were burned. Storm activity and rainfall following the fires increased sediment loads in rivers, resulting in localized fish kills and widespread water-quality deterioration. We collected water samples from the headwaters of the Murray River for sediment and contaminant analysis and assessed changes in water quality using long-term monitoring data. A robust runoff routing model was used to estimate the effect of fire on sediment loads in the Murray River. Peak turbidity in the Murray River reached values of up to 4200 nephelometric turbidity units (NTU), shown as pitch-black water coming down the river. The increase in suspended solids was accompanied by elevated nutrient concentrations during post-bushfire runoff events. The model simulations demonstrated that the sediment load could be five times greater in the first year after a bushfire than in the prefire condition. It was estimated that Lake Hume, a large reservoir downstream from fire-affected areas, would receive a maximum of 600 000 metric tonnes of sediment per month in the period immediately following the bushfire, depending on rainfall. Total zinc, arsenic, chromium, nickel, copper, and lead concentrations were above the 99% toxicant default guideline values (DGVs) for freshwater ecosystems. It is also likely that increased nutrient loads in Lake Hume will have ongoing implications for algal dynamics, in both the lake and the Murray River downstream. Information from this study provides a valuable basis for future research to support bushfire-related policy developments in fire-prone catchments and the mitigation of postfire water quality and aquatic ecosystem impacts. Integr Environ Assess Manag 2021;17:1203-1214. © 2021 Commonwealth of Australia. Integrated Environmental Assessment and Management © 2021 Society of Environmental Toxicology & Chemistry (SETAC).

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.

Exploring the potential of Nelumbo nucifera rhizome on membrane stabilization, mast cell protection, nitric oxide synthesis, and expression of costimulatory molecules.

Immunomodulatory activity of Nelumbo nucifera rhizome was evaluated for its standardized extract (NNRE) with respect to betulinic acid. Various key parameters including erythrocyte membrane stabilization, inhibition of histamine release, reduction in nitric oxide production and depletion of expression of costimulatory molecules of macrophages were estimated. The result displayed that NNRE stabilized erythrocyte membrane significantly at 10 (42.05%) and 100 microg/mL (44.31%). Although considering the protection of mast cells from degranulation, NNRE showed 38.66% (100 microg/mL) and 69.66% (10 microg/mL) degranulation against compound 48/80 (C 48/80). NNRE at 1 and 5 microg/mL inhibited lipopolysaccharide (LPS)-induced activation of macrophages by decreasing the expression of costimulatory molecules. Expression of CD40, CD80, and CD86 by NNRE was seen significantly at 5 microg/mL compared to LPS-treated group. The extracts also inhibited the nitrite concentration at 1 and 5 microg/mL compared to LPS-treated group.

Porin-incorporated liposome induces Toll-like receptors 2- and 6-dependent maturation and type 1 response of dendritic cell.

Porin of Shigella dysenteriae was incorporated in liposome (PIL) and presented to mouse splenic dendritic cells (DC). PIL up-regulated Toll-like receptor (TLR) 2 and TLR6 on DC, showing that co-expression of the two TLRs is involved in recognition of porin. Detection of myeloid differentiating factor 88 (MyD88)-TLR2 complex confirmed interaction between the two for triggering the downstream signaling, which ultimately led to TLR2-dependent nuclear translocation of nuclear factor-kappa B. PIL-induced expression of MHC class II (I-Ab), CD40 and CD80 showed maturation of DC, whereas up-regulation of intercellular adhesion molecule-1 and CCR7 implicated the capacity of splenic DC to migrate. Induction of messenger ribonucleic acid for the chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted indicated a strong bias of PIL for type 1 polarization that was supported by the intracellular expression and release of tumor necrosis factor (TNF)-alpha and IL-12. Along with CD40 and CD80 expression, release of the cytokines of CD11c+ JAWS II cells was inhibited by TLR2 or simultaneous TLR2 and 6 knockdown showing that recognition of PIL by the two TLRs is essential for DC activation and type 1 polarization. The signaling pathway initiated upon recognition of PIL by the TLRs was MyD88 dependent as confirmed by inhibition of IL-6, TNF-alpha and IL-12 release of MyD88-knockdown JAWS II cells. The maturation and polarization of DC induced T(h)1 phenotype, as evident from proliferation, activation and IFN-gamma release of allogeneic CD4+ T cells in response to PIL-stimulated DC, thereby suggesting that the adjuvant activity of PIL can successfully bridge the innate and adaptive immunity.

Priming of CD4+ T cells with porin of Shigella dysenteriae activates the cells toward type 1 polarization.

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive T(h) cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4(+) T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-gamma and had no effect on IL-4 expression. In parallel to the predominant release of the T(h)1 cytokine IFN-gamma, up-regulation of CCR5 on the immune CD4(+) T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1 alpha and regulated on activation normal T cell expressed and secreted showed T(h)1 bias. The porin-primed CD4(+) T cells expressed the mRNA for T-box expressed in T cells, a T(h)1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector T(h)1 cells. The immune CD4(+) T cells proliferated and released IL-2 and IFN-gamma profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4(+) T cells of T(h)1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity.

Vibrio cholerae hemolysin is apoptogenic to peritoneal B-1a cells but its oligomer shepherd the cells for IgA response.

Vibrio cholerae hemolysin (HlyA) can exist as a monomer with hemolytic activity and an oligomer that agglutinates erythrocytes. Biochemical differences accompanying the change in state of aggregation led us to weigh possible differences between the two forms from mucosal immunoregulation perspective. HlyA oligomer-treated murine B-1a cells up-regulated TLR2 and involved the signaling molecules MyD88, TRAF6 and NF-kappaB. The cells subsequently expressed IgM and IgA. HlyA monomer treatment although resulted in TLR2 up-regulation, could not induce these effects. Apoptosis was detected in majority of the monomer-treated cells that involved caspase-9 and caspase-3. This study shows for the first time that two forms of the same protein could drive the host immune cell to two different outcomes, one of death and the other towards activation.

Identification and expression profiling of MSY genes of yak for bull fertility.

Yak (Bos grunniens) is a unique bovine species and considered as lifeline of highlanders. The male subfertility in yak is a matter of concern that causes huge economic loses. The spermatogenesis and male reproduction machinery are critically governed by Y-linked genes which tend to acquire necessary information in the course of evolution. The Y-linked fertility genes are present in multiple copies with testis-limited expression. To understand this novel complexity, 12 male-specific region of Y chromosome (MSY) genes have been studied in the yak. Targeted genes are amplified in male and female genomic DNA and confirmed the male derived specificity. Moreover, testis and sperm-specific expressions of MSY genes are distinct among different tissues. The quantitative polymerase chain reaction results validate the expression pattern of these genes in various tissues with predominant expression intestis and sperm. The sequencing of resultant yak MSY genes gives significant result and shows similarity with cattle (Bos indicus), but few nucleotide mismatches define the proposition of infertile male in the F1 hybrid of cattle and yak. The identified MSY genes can be used to establish male-specific characteristics and to differentiate male and female yak genotypically. Further, these genes may act as valuable resources to understand the capacity of spermatogenesis, embryogenesis, cellular growth, azoospermia and malesubfertility in the yak.

Oligomerization of Vibrio cholerae hemolysin induces CXCR3 upregulation and activation of B-1a cell.

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.

Porin of Shigella dysenteriae activates mouse peritoneal macrophage through Toll-like receptors 2 and 6 to induce polarized type I response.

Porin of Shigella dysenteriae type 1 coexpressed Toll-like receptor (TLR) 2 and TLR6 on peritoneal cavity (PerC) macrophages (MPhi) of C57BL/6 mice implicating that both the TLRs are essential as a combinatorial repertoire to recognize the protein. Besides TLRs, mRNA for MyD88 and TRAF6, and nuclear translocation of NF-kappaB were enhanced that indicate their involvement in tandem in the activity of porin. The protein selectively up-regulated CD80 on the activated MPhi together with MHC class II molecule and CD40, and had no effect on CD86 expression. The porin-induced profile of MIP-1alpha, MIP-1beta and RANTES showed strong bias for chemokines correlated with M1 polarization. Intracellular expression and release of TNF-alpha and IL-12 in presence of porin was found to be TLR2 and NF-kappaB dependent. Induction of TNF-alpha and IL-12 along with the chemokine profile suggests type I polarization of the MPhi that would influence Th1-type response.

Fate and dynamics of metal precipitates arising from acid drainage discharges to a river system.

Neutralisation of acid drainage creates metal-rich precipitates that may impact receiving water bodies. This study assessed the fate of over seven years of acid drainage discharges on the sediments of the lower River Murray (Australia), including the potential for periodic water anoxia to enhance risk via reductive dissolution of amorphous (Fe, Mn and co-precipitated and bound metal) oxide phases. With the exception of one site with restricted water exchange, elevated reducible/reactive metal(oid) (Fe, Ni, As, Co, Zn) concentrations were only observed in the localised wetland-riparian area within approximately 100 m of the discharges. Only a minor exceedance of national sediment quality guideline values occurred for Ni. In the main river channel, elevated reactive metal (Fe, Mn, Ni, Zn) concentrations were also only observed less than approximately 100 m from the drainage discharge point. This appears due to (a) rapid neutralisation of pH leading to metal precipitation and deposition in the localised discharge area, and/or (b) dilution of any metal precipitates entering the main channel with natural river sediments, and/or (c) flushing of precipitates downstream during higher flow conditions. The influence of deoxygenation on metal release was profound with large increases in the concentration of dissolved Fe, Mn, Zn, Ni, and As in the overlying water during laboratory experimental simulations. However, given in situ sediment metal contamination is very localised, it appears on a river reach scale that the acid drainage precipitates will not significantly contribute, over and above, the background release of these metals during these conditions.

Prevalence of paratuberculosis in organized and unorganized dairy cattle herds in West Bengal, India.

AIM: The aim of this study was to determine the prevalence pattern of Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis or Johne's disease, in unorganized as well as organized cattle herds in West Bengal. MATERIALS AND METHODS: Four organized cattle farms with identical management practice in Nadia (n=3) and South 24 Parganas (n=1) districts and three unorganized cattle herds, one each from three districts, namely, Burdwan, North 24 Parganas, and Purba Midnapur, were selected randomly and screened for paratuberculosis by delayed-type hypersensitivity (DTH) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 191 animals tested by DTH, 57 (29.8%) were found to be positive in comparison to 72 (37.7%) by ELISA. In organized farms, seropositivity varied from 13.3% to 53.1%, whereas in unorganized sector, it ranged from 5% to 6.7% with one area having exceptionally high prevalence, i.e. 53.3%. The range of positivity detected by DTH both in organized farms and backyard sectors varied from 0% to 46.7%. By employing both DTH and ELISA together, the positivity of animals in organized and unorganized herds was 19.9% and 8%, respectively. CONCLUSION: The results indicate that animals in organized farms are much more prone to paratuberculosis than others. For screening the herd, both DTH and ELISA should be used simultaneously to increase the test sensitivity in order to minimize its further spread adopting control programs.

Pathogen-associated porin turns IL-10 competent B-1a cells toward proinflammatory cytokine response.

Shigellosis is a major problem in the developing countries causing mortality and morbidity particularly among the children. Shigella spp. harbours the epithelial cells of the human colon to infect the host and spread the disease. We analyzed the response of B-1a cells, the major component of the mucosal immune system to porin of Shigella dysenteriae type 1. We show that porin while proliferating B-1a cells, deplete Siglec-G, the inhibitory molecule present on B-1a cells. Adjuvanticity of porin has been shown to govern innate signaling for promoting host adaptive immune response. Up-regulation of CD69 and CD40 denotes activation of the cells parallel to abrogation of Siglec-G. As a result of cell activation, porin stimulated the inflammatory cytokines of CD5+ B-1a cells, otherwise rich in IL-10. The work shows B-1a cell responses promote the immunopotentiating activity of porin.

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.

Up-regulation of CD80-CD86 and IgA on mouse peritoneal B-1 cells by porin of Shigella dysenteriae is Toll-like receptors 2 and 6 dependent.

Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in robust NF-kappaB activation for signaling. Porin treatment of B-1 cell populations of C57BL/6 mice, and C3H/HeJ mice in particular, selectively up-regulated the expression of the costimulatory molecules. CD80 expression got enhanced on the B-1a cells whereas CD86 got solely expressed on B-1b cells. Porin-induced cell surface expression of IgM and IgA on B-1 cell populations from C57BL/6 mice. The IgA-generating capacity, hallmark of mucosal immune response, was confirmed with B-1 cells of C3H/HeJ, the lipopolysaccharide non-responder mouse, in response to the protein. The porin-mediated induction of IgA was augmented by interleukin-6 on B-1a and B-1b cells, by 2.4- and 2.6-fold, respectively. The IgA expressed on both B-1a and B-1b cell surfaces after 72 h of culture was found to bind to the 38 kDa monomer of porin confirming it to be anti-porin IgA antibody.