EDITORIAL article Front. Cell. Infect. Microbiol., 18 May 2023Sec. Clinical Microbiology Volume 13 - 2023 | https://doi.org/10.3389/fcimb.2023.1216183

Supplementary Figures 1-2, Tables1-3 from Reduced Colitis-Associated Colon Cancer in <i>Fat-1</i> (<i>n</i>-3 Fatty Acid Desaturase) Transgenic Mice

Supplementary Figures 1-2, Tables1-3 from Reduced Colitis-Associated Colon Cancer in Fat-1 (n-3 Fatty Acid Desaturase) Transgenic Mice

The emerging obesity epidemic and its co-morbid conditions, such as colorectal cancer, pose a great challenge to global health and healthcare cost. This reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in individuals with obesity. Extensive epidemiological studies have demonstrated a consistent and compelling association between obesity and the risk of cancer development and progression. Furthermore, our previous studies demonstrate that obesity induced by high-fat diet results in the expansion of the colonic stem cell pool during cancer initiation. However, the mechanistic link between obesity and the initiation and development of colon cancer remains elusive. Since the biophysical properties of the plasma membrane contribute to cellular signaling, we hypothesized that obesity alters colonic stem cell homeostasis via modulation of the biophysical properties of the plasma membrane, subsequently altering cell signaling events. Our studies utilized complementary in vivo and ex vivo mouse models including colonic organoids. Plasma membrane biophysical properties and cholesterol content were determined utilizing the physico-chemical sensitive dye, Di-4-ANEPPDHQ and Filipin III, respectively. Super-resolution stochastic optical reconstruction microscopy (STORM) was utilized to determine the nanoscale clustering of Wnt receptor, LRP6, and epidermal growth factor receptor (EGFR). Effects of nanoclustering on the activation status of downstream signaling targets was subsequently assessed. High-fat diet induced obesity increased plasma membrane free cholesterol and rigidity of colonic crypts and Lgr5+ cells. The increase in rigidity was associated with an increase in the nanoclustering of key stem cell regulating membrane-bound receptors, LRP6 and EGFR. These biophysical perturbations were associated with changes in stem cell signaling and organoid-forming efficiency, a measure of stemness. Our results indicate that obesity alters the biophysical properties of the plasma membrane which influences the clustering and downstream signaling of membrane receptors. These findings are noteworthy because they may explain in part how obesity drives colon cancer progression. This work was supported by NIH grant R35CA197707.

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5+ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

Globally, an estimated 16 million people are living with tuberculosis (TB). Nutritional support was the mainstay of TB treatment before the advent of anti-TB drugs. Due to the difficulty of curing patients with antimicrobial drug resistance, advanced TB disease, and comorbidities, there is renewed interest in nutritional support in the management of TB. Undernutrition is an important risk factor for developing TB. TB causes anorexia, weight loss and cachexia which have been associated with a specific cytokine milieu. Malnutrition also correlates strongly with disease severity; therefore, normalization of body weight in response to treatment is positive sign. The goal of nutritional interventions is to compensate for the effects of TB on metabolism and the increased activity of the immune system. A careful assessment of nutritional status should be performed before assigning a supplementation regimen although a simplified, more generic approach may be more suitable in some circumstances. Nutritional support must be adapted to each geographic region and socioeconomic context. Supplementation of multiple micronutrients during treatment has been associated with reduced mortality regardless of HIV status. Due to its interference with vitamin B6 metabolism, isoniazid should be administered with vitamin B6 supplements.

Doce enfermos con tuberculosis pulmonar confirmada que reperidamente tuvieron reacciones negativas a pruebas cutáneas con 250 UI de proteína pura derivada (PPD) de tuberculina, se estudairon utilizando puebas de inmunidad celular in vivo e in vitro. ÚNicamente 2 pacientes respondieron a las pruebas cutáneas con candidina e histoplasmina y solo uno se pudo sensibilizar con aplicaciones tópicas de dimitroclorobenceno (DNCB). En estos individuos anérgicos la respuesta blastogénica de los linfocitos de sangre periférica, al cultivarlos con fitohemaglutinina, mitógeno de hierba carmín (pokeweed) y PPD fue significativamente menos que la de los linfocitos de los pacientes controles con reacciones positivas a tuberculina.

Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4(+) T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4(+) T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4(+) T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4(+) T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

Undernutrition is an important risk factor for the development of tuberculosis (TB). Understanding the link between undernutrition and susceptibility to TB is based on a conceptual model for the transmission and pathogenesis of TB. The risk of progression from infection to disease increases substantially in undernourished individuals which is likely due to the detrimental effects of undernutrition on cell-mediated immunity (CMI, essential for the control of mycobacterial growth). Evidence in humans linking undernutrition to TB risk is indirect and surprisingly weak from the perspective of scientific rigor. The majority of evidence in humans comes from a large body of uncontrolled observations and ecological studies in which the effects of undernutrition cannot be separated from the effects of socioeconomic disparities. While cross-sectional and case–control studies help to support an association between undernutrition and development of TB, they do not address the temporal relationship. Only two cohort studies have examined the temporal relationship between micronutrients and TB incidence. Experimental animal models have allowed scientists to elucidate causal links between nutritional deficiencies, immune system function, and TB. Although TB is clearly related to undernutrition, the risk relative to specific levels and types of protein-energy deficiency and micronutrient deficiencies remains to be defined.

Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms.

Background: Dietary factors such as high-sodium or high-fat (HF) diets have been shown to induce a proinflammatory phenotype. However, there is limited information with respect to how microenvironments of distinct intra-abdominal adipose depots respond to the combination of a high-salt, HF diet. Objective: We tested the hypothesis that HF feeding would cause changes in distinct adipose depots, which would be further amplified by the addition of high salt to the diet. Methods: Twenty-sevenmale C57BL6mice were fed an HF diet (60% of kcal from fat), an HF + high-salt diet (4%wt:wt), a control diet [low-fat (LF);10% of kcal from fat], or an LF + high-salt diet for 12 wk. The main sources of fat in the diets were corn oil and lard. Adipokines in serum and released from adipose tissue organ cultures were measured by immunoassays. QIAGEN’s Ingenuity Pathway Analysis was used to perform functional analysis of the RNA-sequencing data from distinct adipose depots. Results:Diet-induced obesity resulted in a classical inflammatory phenotype characterized by increased concentrations of circulating inflammatory mediators (38–56%) and reduced adiponectin concentrations (27%). However, high-salt feeding did not exacerbate the HF diet–induced changes in adipokines and cytokines. Leptin and interleukin-6 were differentially released from adipose depots and HF feeding impaired adiponectin and resistin secretion across all 3 depots (34–48% and 45–83%, respectively). The addition of high salt to the HF diet did not further modulate secretion in cultured adipose tissue experiments. Although gene expression data from RNA sequencing indicated a >4.3-fold upregulation of integrin aX (Itgax) with HF feeding in all 3 depots, markers of cellular function were differentially expressed in response to diet across

BACKGROUND Dietary factors such as high-sodium or high-fat (HF) diets have been shown to induce a proinflammatory phenotype. However, there is limited information with respect to how microenvironments of distinct intra-abdominal adipose depots respond to the combination of a high-salt, HF diet. OBJECTIVE We tested the hypothesis that HF feeding would cause changes in distinct adipose depots, which would be further amplified by the addition of high salt to the diet. METHODS Twenty-seven male C57BL6 mice were fed an HF diet (60% of kcal from fat), an HF + high-salt diet (4% wt:wt), a control diet [low-fat (LF);10% of kcal from fat], or an LF + high-salt diet for 12 wk. The main sources of fat in the diets were corn oil and lard. Adipokines in serum and released from adipose tissue organ cultures were measured by immunoassays. QIAGEN's Ingenuity Pathway Analysis was used to perform functional analysis of the RNA-sequencing data from distinct adipose depots. RESULTS Diet-induced obesity resulted in a classical inflammatory phenotype characterized by increased concentrations of circulating inflammatory mediators (38-56%) and reduced adiponectin concentrations (27%). However, high-salt feeding did not exacerbate the HF diet-induced changes in adipokines and cytokines. Leptin and interleukin-6 were differentially released from adipose depots and HF feeding impaired adiponectin and resistin secretion across all 3 depots (34-48% and 45-83%, respectively). The addition of high salt to the HF diet did not further modulate secretion in cultured adipose tissue experiments. Although gene expression data from RNA sequencing indicated a >4.3-fold upregulation of integrin αX (Itgax) with HF feeding in all 3 depots, markers of cellular function were differentially expressed in response to diet across depots. CONCLUSION Collectively, these findings highlight the role of distinct adipose depots in mice in the development of obesity and emphasize the importance of selecting specific depots to study the effects of therapeutic interventions on adipose tissue function.

n-3 polyunsaturated fatty acids (PUFA) have been shown in many clinical studies to attenuate inflammatory responses. Although inflammatory responses are orchestrated by a wide spectrum of cells, CD4+ T cells play an important role in the etiology of many chronic inflammatory diseases such as inflammatory bowel disease and obesity. In light of recent concerns over the safety profiles of non-steroidal anti-inflammatory drugs (NSAIDs), alternatives such as bioactive nutraceuticals are becoming more attractive. In order for these agents to be accepted into mainstream medicine, however, the mechanisms by which nutraceuticals such as n-3 PUFA exert their anti-inflammatory effects must be fully elucidated. Lipid rafts are nanoscale, dynamic domains in the plasma membrane that are formed through favorable lipid–lipid (cholesterol, sphingolipids, and saturated fatty acids) and lipid–protein (membrane–actin cytoskeleton) interactions. These domains optimize the clustering of signaling proteins at the membrane to facilitate efficient cell signaling which is required for CD4+ T cell activation and differentiation. This review summarizes novel emerging data documenting the ability of n-3 PUFA to perturb membrane-cytoskeletal structure and function in CD4+ T cells. An understanding of these underlying mechanisms will provide a rationale for the use of n-3 PUFA in the treatment of chronic inflammation.

The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.

Some aspects of the early development of tuberculous infection were compared in vaccinated and nonvaccinated guinea pigs after low dose, respiratory challenge with Mycobacterium tuberculosis H37Rv. Changes with time of the logarithm of the number of viable mycobacteria recovered from the lungs of animals from both groups sacrificed at several intervals between zero and 35 days after challenge described a sigmoid curve with three phases: a lag phase, a phase of logarithmic growth, and a stationary or decline phase. The influence of vaccination on the rate of accumulation of the challenge organism was not detected before 10 to 14 days after infection. The significance of these results is discussed with reference to the important role a critical amount of antigen can play in any test system in which acquired cellular immunity is studied.

13 n-3 PUFA have been shown in many clinical studies to attenuate inflammatory responses. 14 Although inflammatory responses are orchestrated by a wide spectrum of cells, CD4 T cells 15 play an important role in the etiology of many chronic inflammatory diseases such as 16 inflammatory bowel disease and obesity. In light of recent concerns over the safety profiles of 17 non-steroidal anti-inflammatory drugs (NSAIDs), alternatives such as bioactive nutraceuticals 18 are becoming more attractive. In order for these agents to be accepted into mainstream 19 medicine, however, the mechanisms by which nutraceuticals such as n-3 polyunsaturated fatty 20 acids (PUFA) exert their anti-inflammatory effects must be fully elucidated. Lipid rafts are 21 nanoscale, dynamic domains in the plasma membrane that are formed through favorable lipid22 lipid (cholesterol, sphingolipids, and saturated fatty acids) and lipid-protein (membrane-actin 23 cytoskeleton) interactions. These domains optimize the clustering of signaling proteins at the 24 membrane to facilitate efficient cell signaling which is required for CD4 T cell activation and 25 differentiation. This review summarizes novel emerging data documenting the ability of n-3 26 PUFA to perturb membrane-cytoskeletal structure and function in CD4 T cells. An 27 understanding of these underlying mechanisms will provide a rationale for the use of n-3 PUFA 28 in the treatment of chronic inflammation. 29

There is an urgent need to elucidate the mechanistic links between obesity and colon cancer. Convincing evidence for the role of Lgr5(+) stem cells in colon tumorigenesis has been established; however, the influence of obesity on stem cell maintenance is unknown. We assessed the effects of high fat (HF) feeding on colonic stem cell maintenance during cancer initiation (AOM induced) and the responsiveness of stem cells to adipokine signaling pathways. The number of colonic GFP(+) stem cells was significantly higher in the AOM-injected HF group compared to the LF group. The Lgr5(+) stem cells of the HF fed mice exhibited statistically significant increases in cell proliferation and decreases in apoptosis in response to AOM injection compared to the LF group. Colonic organoid cultures from lean mice treated with an adiponectin receptor agonist exhibited a reduction in Lgr5-GPF(+) stem cell number and an increase in apoptosis; however, this response was diminished in the organoid cultures from obese mice. These results suggest that the responsiveness of colonic stem cells to adiponectin in diet-induced obesity is impaired and may contribute to the stem cell accumulation observed in obesity.